text:
A novel composite scaffold of Cu-doped nano calcium-deficient hydroxyapatite/multi-(amino acid) copolymer for bone tissue regeneration
Authors Mou P, Peng H, Zhou L, Li L, Li H, Huang Q
Received 13 December 2018
Published 7 May 2019 Volume 2019:14 Pages 3331—3343
Editor who approved publication: Dr Linlin Sun
Ping Mou,1 Haitao Peng,2 Li Zhou,3 Lin Li,2 Hong Li,2 Qiang Huang1
1Department of Orthopedic Surgery, West China Hospital, West China Medical School, Sichuan University; 2School of Physical Science and Technology, Sichuan University; 3Core Facility of West China Hospital, Sichuan University, Chengdu, Sichuan Province 610041, People's Republic of China
Background and methods: A Cu-doped composite scaffold of nano calcium-deficient hydroxyapatite (n-CDHA)/multi(amino acid) copolymer (MAC) was prepared. The structure, porosity, morphology and compressive strength of the scaffolds were characterized, the in vitro degradability in phosphate-buffered solution (PBS) and cell responses to the scaffolds were investigated, and in vivo stimulation of bone formation were analyzed.
Results: The scaffolds showed the compressive strength of approximately 12 MPa and total porosity of about 81%. Weight loss of the composite scaffolds was 63% after 16-week immersion in PBS. Cu release in scaffolds showed a marked dependence on the initial amount in the scaffolds over time. Cu-doped n-CDHA/MAC scaffolds with the content of Cu 0.5% and 1% in mass ratio showed better cell responses to proliferation and differentiation of rat bone marrow stromal cells (rBMSCs) than that with no Cu. After 12-week implantation in rabbits, 1% Cu-doped n-CDHA/MAC showed better ability of angiogenesis and osteogenesis compared to 0% Cu-doped n-CDHA/MAC.
Conclusion: The 1% Cu-doped n-CDHA/MAC composite scaffold showed good capacity of angiogenesis and osteogenesis, and the Cu showed positive effects on cell growth and osteogenesis. And it has potential to be used as bone regeneration scaffolds.
Keywords: calcium-deficient hydroxyapatite, multi-(amino acid) copolymer, copper, osteogenesis, degradability, cytocompatibility
Osteonecrosis of the femoral head (ONFH) associated with alcohol abuse and/or corticosteroids1 is a potentially devastating disease due to interruption of circulation that limits the transmission of oxygen and nutrients to femoral head. During the last several decades, early-stage joint-preserving operative techniques have been used to slow the progression of ONFH,2,3 which include core decompression, femoral osteotomy, nonvascularised bone grafting (NVBG), vascularized bone grafting (VBG) and cell-based treatment.4,5
Although core decompression can reduce the intraosseous hypertension resulting from swelling and infiltration of cells due to the progressive necrosis and provide symptomatic relief especially in hips with symptomatic, smaller-sized, pre-collapse necrosis,6 no obvious relief could be found when the femoral head collapsed. In addition, core decompression can be in combination with tantalum rods, bone substitutes, arthroscopy, and cell treatment. However, there are still debates on the results of the techniques between Western and East Asian countries.4 Femoral osteotomy allows healing and postpones progression by moving the necrotic region from weight-bearing area and shows favourable results.7–9 However, the complications including poor fixation and/or malposition, malunion after osteotomy are high-incidence5,10 as well as more difficult to switch to total hip arthroplasty (THA) if an osteotomy fails.11 NBVG can support the femoral head structurally and decompress the necrotic region to facilitate healing and remodelling of femoral head, but using autogenous cancellous grafting can prolong surgical time and cause additional pain of donor site, while the antigenicity and potential danger of spreading diseases for allograft implants.12,13 Although VBG can provide structural support and restore blood supply of live bone,14 yet technical difficulties and prolonged operation time are required. Also, current studies on VBG do not have a control group and are not randomized. So, there is no consensus on the true effect on the progression of osteonecrosis.4 Osteonecrosis results from lack of cells which can promote osteogenesis. So, cell therapy to potentially regenerate bone was considered.15 A recent systematic review15 concluded that cell therapy was safe for osteonecrosis and could improve clinical outcomes. Nonetheless, the authors15 suggest strict application indications for osteonecrosis should be determined and standardization of cell therapy should be established before cell-based treatment adopted. In brief, the current operative methods to treat early-stage ONFH just provide the mechanical support of necrotic region and there is no consensus on the true results of VBG on the progressive osteonecrosis. So, how to synthesize material combining osteogenesis with angiogenesis to induce bone regeneration and vascularization is significant to cure early-stage ONFH.
Angiogenesis is essential in bone regeneration because blood vessels transport oxygen and nutrients to regenerated bone, which are crucial for bone regeneration and defect restoration. Copper (Cu), a vital trace element, could stimulate osteogenesis and angiogenesis of mesenchymal stem cells (MSCs).16–18 Simultaneously, the research19 has demonstrated that in culture medium, even the concentration of Cu reached up to 250 µM, there is no adverse and side effects for cells for continuous culture of 10 days, which shows the biosecurity of Cu in regions. And literature also demonstrated that doping Cu into biomaterials to form novel scaffolds with characteristic of slow-release of Cu could enhance their original angiogenesis and osteogenesis properties.20
Moreover, according to Li's research,21 nano calcium-deficient hydroxyapatite/multi-(amino acid) copolymer (n-CDHA/MAC), an eligible degradable composite bone substitute biomaterial, consisting of inorganic n-CDHA and an amide linkage copolymer composed of 6-aminocaproic acid and other five amino acids, not only was provided with good mechanical performance, but also outstanding osteogenesis when the n-CDHA occupies 40 wt% of n-CDHA/MAC. And n-CDHA/MAC presented macropores with the size from 100 to 600 um and porosity of 81%, which were similar to the reported literature showing satisfactory bone ingrowth and mineralized bone regeneration.22 Furthermore, the previous researches19,21,23,24 has demonstrated that n-CDHA/MAC possess excellent mechanical strength, degradability and bioactivity.
Therefore, on account of the properities n-CDHA/MAC and Cu respectively, we synthesized a novel composite bone substitute scaffold: Cu-doped n-CDHA/MAC with functional properties including osteogenesis and angiogenesis. To our knowledge, this is the first study research Cu-doped n-CDHA/MAC for bone regeneration. So, the aim of present study is to investigate the biosecurity of the scaffolds as the foundation of the treatment of ONFH. In particular, the physical and chemical properties, the ability of angiogenesis and osteogenesis were evaluated.
Synthesis of Cu-doped n-CDHA/MAC composite
The Cu-doped n-CDHA slurry was prepared in a reactor under 40℃ according to a precipitation route described elsewhere.25 Briefly, a certain amount of CuSO4·5H2O (AR, Guang dou Guanghua Sci-Tech co., Ltd) and 10 mole calcium nitrate tetrahydrate (Ca (NO3)2.4H2O, 99.2% Aldrich) was added dropwise to 6 mole diammonium hydrogen phosphate ((NH4)2HPO4, 99.2% Aldrich), and the pH was continuously controlled and adjusted at 9.0±0.1 by adding NH4OH (AR, Chengdou kelong Chemical Reagent Co., Ltd). Precipitate was aged in mother solution for 48 h, washed with NH4OH aqueous solution and alcohol (AR, Sichuan Xilong Chemical co., Ltd). Cu-doped n-CDHA with various Cu/CDHA ratio (W/W, 1.25%, 2.5%, 5%, and 12.5%) were obtained.
The preparation method of Cu-doped n-CDHA/MAC composite with 40 wt% Cu-doped n-CDHA was described in detail in the reference,21 and the scaffold was prepared according to the reference.26 The Cu in the obtained scaffolds were 0.5%, 1%, 2% and 5%, respectively (Figure1).
Figure 1 A flow chart about the preparation of scaffold.Abbreviations: n-CDHA, nano calcium-defcient hydroxyapatite; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; PAA, polyamino acid.
Cu, porosity and morphology of the scaffolds
The concentrations of Cu in scaffolds were detected by inductively coupled plasma mass spectrometry (ICP, IRIS 1,000, Thermo Electron Corporation, Waltham, MA, USA). The densities of different Cu-doped n-CDHA/MAC composite scaffolds (φ6×12 mm), ρa, were measured by the ratio of mass and volume. The skeletal densities, ρd, was also measured with compact samples (φ6×12 mm). The pyknotic samples were handled by the same process of Cu-doped n-CDHA/MAC composite scaffolds without foaming agent, and the total porosity was calculated by the formula: Pt(%) =(1-ρa/ρd)×100%. Three replicates were done for each group, and the results were presented as mean ± SD.
Also, the morphology of Cu-doped n-CDHA/MAC composite scaffolds was shown by Micro-CT (PerkinElmer, Quantum GX, America).
Compressive strength measurement
The compressive strengths of different Cu-doped n-CDHA/MAC composite scaffolds were handled by mechanical testing machine (REGER 30–50, Shenzhen Reger Co., Ltd. China) with 50 kN load. The cross-head speed was 1 mm/min, and the load was applied until the samples (φ6×12 mm) were compressed to approximately 50% in height or the samples collapsed. Three replicates were done for each group, and the results were expressed as mean ± SD.
In vitro degradability and cu release in PBS
The in vitro degradability of Cu-doped n-CDHA/MAC composite scaffolds was performed by soaking the scaffold samples (φ6×12 mm) into phosphate-buffered saline (PBS, pH=7.4) for 16 weeks and was evaluated at different time points. We washed scaffold samples, dried them at 80 °C in vacuum oven for 4 h and weighed them the initial weight (Wi). The PBS was equipped with a solid/liquid ratio of 1 g/50 ml in polyethylene (PE) tubes, which were capped and placed in a shaking water bath (37±0.5 °C and 72 rpm). At 1, 2, 4, 8, 12 and 16 weeks, the samples were washed and dried, and the residual weight (Wt) were measured. The weight loss ratio was tested according to the following formula: Weight loss = (Wi-Wt)/Wi ×100%. Simultaneously, the content of Cu in the soaking solution were determined by by inductively coupled plasma mass spectrometry (ICP, IRIS 1000, Thermo Electron Corporation, Waltham, MA, USA) and the release content of Cu in vitro was obtained.
Rat bone mesenchymal stem cells (rBMSCs) (ATCC; Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) equipped with 10% fetal bovine serum (FBS) plus 1% (v/v) Penicillin-Streptomycin solution (Gibco, Life Technologies, USA) at 37 °C and CO2 5% in a humidified atmosphere. Cells were incubated in 25 cm2 flasks to reach 80% confluence and then separated for further experiments. Different Cu-doped n-CDHA/MAC composite scaffold samples with the size of φ6×2 mm were used for cell experiments, and tissue culture plate (TCP) was used as a control. These samples were sterilized by ethylene oxide gas.
To investigate cell adhesion on the scaffolds, 1 ml rBMSCs suspension with a density of 8×104 cells/ml were seeded onto Cu-doped scaffolds and located in 24-well TCPs. The cells and samples were cultured at 37 °C and a humidified 5% CO2 atmosphere for 1 and 4 days, after washing for three times with PBS, cells and samples were fixed in 4% glutaraldehyde for 24 h. The specimens were then dehydrated in ascending concentrations of ethanol for 15 min. Later, the samples were critical point dried, and the morphology of the cells at 1 and 4 days was shown using SEM (Hitachi S-450, Japan).
BMSC proliferation was assessed by cell counting kit 8 (CCK8, KeyGEN BioTECH, Nanjing, China) following the manufactory's instructions. Briefly, the cells were attached to the wall after cultivating for 24 h. Then the medium was added material leaching solution (preparation of the material leaching solution: the materials were immersed in the culture medium for 48 h under the environment of 37 °C, and then the medium was filtered through filter membrane with diameter of 0.22 micron to obtain it). The medium used by the blank control group was kept unchanged. And the medium was changed every 2 days. Add 10 µl CCK8 into the wells after cultivating 1,4,7 days and cultivate for 2 h at 37 °C, 5% (V/V) thermostatic cultivation in CO2 saturated humidity. The absorbance was measured at 450 nm using a microplate reader (Multiskan MK3, Thermo Electron Corporation, Waltham, MA, USA).
Alkaline phosphatase activity
For osteogenic differentiation of rBMSCs, cells were cultured in osteogenic medium (basal medium additionally provided with 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid and 100 nM dexamethasone). And rBMSCs were seeded on the scaffold samples and alkaline phosphatase (ALP) activity of cells was measured at 1, 4 and 7 days to evaluate the effect of Cu-doped n-CDHA/MAC on the early osteogenic differentiation. Medium was changed up every two days. Briefly, we aspirated the culture medium, added 200 μl of 1% Nonidet P-40 (NP-40) solution to each well and incubated for 1 h at 37 °C. After the cell lysate was centrifuged, 50 μl supernatant was added to 96-well plates. 50 μl, 2 mg/ml P-nitrophenyl phosphate (Sangon, Shanghai, China) substrate solution including 0.1 mol/L glycine and 1 mmol/L MgCl2·6H2O was added and incubated for 30 min at 37 °C. At last, we added 100 μl 0.1 N NaOH to quench the reaction. The absorbance of ALP at λ=405 nm was quantified on a microplate reader (SPECTRAmax 384, Molecular Devices, USA). The total protein content was measured by the bicinchoninic acid protein assay kit (Pierce Biotechnology Inc, Rockford, IL). The absorbance was read at λ=405 nm and the total protein was estimated. The ALP levels were obtained as the changed OD values and normalized to the total protein content.
Animal experiment
The animal experiments were carried out in Sichuan University and the protocol was approved by the Biomedical Ethics Committee of West China Hospital, Sichuan University (2017046A). During the whole animal experiments, laboratory animal guideline for ethical review of animal welfare for the animal was followed according to National Standard of China. 11 mature New zealand white rabbits (2.0–2.5 kg) were operated to confirm the role of scaffolds in bone repair. After skin preparation and anesthesia, a 2 cm incision in the lateral distal femur was performed to expose the femoral condyle. A cavitary defect of 4 mm in diameter and 8 mm in depth was made by special bone drill. Saline irrigation was used to wash the wounds, the blank group, 0% Cu-doped and 1% Cu-doped n-CDHA/MAC composite scaffolds were implanted into the bone defects. To prevent infection, each rabbit received penicillin intramuscularly 10,000 U/kg, one preoperative dose and two postoperative doses. At 4, 8 and 12 weeks after surgery, the animals were sacrificed by injecting overdose anesthetics and the samples were utilized for imaging examinations and histological analysis.
To observe the process of bone formation and degradation of scaffolds, some samples were examined using X-ray radiograph at 4, 8 and 12 weeks after implantation.
After 4, 8 and 12 weeks, the rabbits were sacrificed for histological analysis. The implants were harvested and immediately fixed in 10% buffered formaldehyde solution for one week. After that, the samples were decalcified for one month and embedded in paraffin. Thin sections were made and then stained with routine Hematoxylin and eosin (HE) staining.
Data are presented as mean ± SD. Statistical difference was analyzed using one-way analysis of variance and Tukey's multiple comparison tests. A value of P<0.05 was considered to be statistically significant.
Physicochemical characteristics of composite scaffolds
The morphology of Cu-doped n-CDHA/MAC composite scaffold was observed by Micro-CT. It was found that the composite scaffolds showed densely distributed macropores with the diameter from 100 µm to 500 µm. And the macropores were interconnected by little pores located in the walls of macropores (Figure 2).
Figure 2 Micro-CT images of 1% Cu-doped n-CDHA/MAC with porosity of 81%. (A) cross-section of scaffold; (B) three-dimensional reconstruction of scaffold. The red arrow represents the interconnected pores located in the walls of macropores.Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.
The total porosity changed little along with different ratio of Cu (Table 1). It could also been found that the compressive strength of Cu-doped n-CDHA/MAC composite scaffolds almost keep the same regardless of the variation of Cu concentration (Table 1). Besides, the concentrations of Cu in scaffolds were also shown in Table 1.
Table 1 Total porosity, compressive strength and concentration of Cu in scaffolds of Cu-doped n-CDHA/MAC composite scaffolds
Weight loss of scaffolds in PBS
It also showed the weight loss of different Cu-doped n-CDHA/MAC composite scaffolds soaked in PBS over time. The total weight loss of samples increased in PBS with time, demonstrating that the composite scaffolds were degradable. Moreover, for different Cu-doped scaffolds, it was demonstrated that they showed similar degradation rate, indicating that Cu in composite had little influence on scaffolds. Also, the composite scaffolds exhibited high degradation rate at the first 4 weeks with weight loss greater than 8% every week, and then gradually decreased. At the end of the test, the overall degradation of material (weight loss) reached almost 63% (Figure 3). During the the whole process, all samples were gradually degraded and no collapse of samples were observed visually, indicating the samples could maintain its strength during degradation and meet the requirement of mechanical support for bone regeneration.
Figure 3 Weight loss of n-CDHA/MAC composite scaffolds with different concentration of Cu after immersing in PBS over time.Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; PBS, phosphatic buffer solution.
Cu release of scaffolds in PBS
Cu release of different scaffolds were obtained. The concentrations of Cu in PBS increased with the increase of Cu in the scaffolds. And it was also found that the contents of Cu released from scaffolds decreased with time, indicating the consistent tendency with the weight loss of scaffolds. The composite scaffolds showed high Cu release at the first 4 weeks and then gradually decreased. After soaking 1, 2, 4, 8, 12 and 16 weeks, the concentrations of Cu of 0.5% Cu-doped n-CDHA/MAC were 1.6 ppm,1.3 ppm,1.1 ppm,0.9 ppm,0.3 ppm and 0 ppm, respectively. The concentrations of Cu of 1% Cu-doped n-CDHA/MAC were 2.3 ppm,2.5 ppm,2.1 ppm, 1.5 ppm,0.9 ppm and 0.4 ppm, respectively. The concentrations of Cu of 2% Cu-doped n-CDHA/MAC were 5 ppm,4.3 ppm,4.1 ppm,3.6 ppm,1.8 ppm and 0.9 ppm, respectively. And the concentrations of Cu of 5% Cu-doped n-CDHA/MAC were 11.5 ppm,11 ppm,10.5 ppm,9.6 ppm,5.9 ppm and 2.2 ppm, respectively (Figure 4). It could be seen that the release of Cu from the scaffolds into PBS showed a marked dependence on the weight loss and initial amount of Cu in the scaffolds.
Figure 4 Cu release curve of different Cu-doped n-CDHA/MAC scaffolds in PBS. Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; PBS, phosphatic buffer solution.
Cell adhesion and proliferation
SEMs of rBMSCs attached on the surfaces of Cu-doped n-CDHA/MAC composite scaffolds were performed at 1 and 4 days (Figure 5). It was found that the cells attached well to the scaffold surfaces and the cells were well spread on the surfaces of scaffolds after cultivating for 1 days. Besides, the cells also developed pseudopods, forming close attachment between the cells and scaffold surfaces. 1% Cu-doped n-CDHA/MAC showed the best cell adhesion with more pseudopods, indicating the best cytocompatibility of 1% Cu-doped composite scaffold. At 4 days, it was shown that cells attached to the composite scaffolds increased. the cells spread out and formed a cellular layer with close attachment to the scaffold surfaces. And more cells could be found attached on 1% Cu-doped composite scaffold surface. And it was demonstrated that Cu could promote cell adhesion and enhance the biological properties of composites.
Figure 5 SEM micrographs of rat BMSCs cells attachment on the surfaces of Cu-doped n-CDHA/MAC composite scaffolds after 1 days (A, C, E, G, I) and 4 days (B, D, F, H, J) of culture. (A and B), 0% Cu-doped n-CDHA/MAC; (C and D), 0.5% Cu-doped n-CDHA/MAC; (E and F), 1% Cu-doped n-CDHA/MAC; (G and H), 2% Cu-doped n-CDHA/MAC; (I and J), 5% Cu-doped n-CDHA/MAC.Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer; SEM, scanning electron microscopy; BMSCs, bone marrow stromal cells.
The CCK8 was conducted to continuously measure the proliferation of rBMSCs cultured on different Cu-doped scaffolds at 1, 4 and 7 days because optimal density (OD) values could act as a direct indicator for cell proliferation on scaffolds. A gradually increasing tendency of cell proliferation in a time-dependent manner was found in each group, although there was no statistically difference in proliferation rate at 1 day. After cultivating for 4 days, obvious differences were found in experimental group compared to control group (P<0.05, respectively). The 0%, 0.5% and 1% Cu-doped group showed superior proliferation to control group, while 2% and 5% Cu-doped group presented inferior proliferation to control group. And at 7 days, the differences were still obvious and statistically significant differences were also found in 0.5%, 1%, 2% and 5% compared to control group (P<0.05, respectively) (Figure 6). For 1% Cu-doped group, the most significant increase was observed compared to other groups. Also, for 2% and 5% Cu-doped group, it showed a conspicuous decrease compared to control group, which indicated cytotoxicity to the rBMSCs. So, these results confirmed the optimal Cu-doped n-CDHA/MAC to promote cell proliferation was 1% in mass ratio and when this ratio was more than 2%, it would show toxic effects to rBMSCs.
Figure 6 CCK assay showing the cell growth of rat BMSCs cultured with different Cu-doped n-CDHA/MAC composite scaffolds at day1, day 4 and day 7. * means significant difference between Cu-doped groups and control group (TCP) (p<0.05). Abbreviations: CCK, cell count kit; BMSCs, bone marrow stromal cells; TCP, tissue culture plate; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.
ALP activity
ALP is a specific indicator of osteogenic differentiation. ALP activity of rBMSCs cultured on the composite scaffolds was assessed at 1, 4 and 7 days. The ALP activity increased with the extension of time for five different composite scaffolds. Statistically difference was found in 1% Cu-doped group compared to 0% Cu-doped group after cultivating for 1 day (P=0.04). Moreover, at 4 days, obvious difference was found in 1%, 2% and 5% Cu-doped groups compared to 0% Cu-doped group (P<0.001, P=0.012 and P=0.001, respectively). And at 7 days, the differences became more obvious and statistically significant differences were found among groups (Figure 7). For 1% Cu-doped group, a significant increase was observed compared to 0% Cu-doped group. Also, for 2% and 5% Cu-doped group, it showed an obvious decrease compared to 0% Cu-doped group. All tested scaffolds containing Cu ranged from 0% to 1% of n-CDHA/MAC showed an increased mineralization of the cells, indicating that the scaffolds with proper content of Cu can stimulate the osteoblastic differentiation of rBMSCs.
Figure 7 ALP activity of rat BMSCs cultured on different Cu-doped n-CDHA/MAC composite scaffolds at day 1, day 4 and day 7. * means significant difference between Cu-doped groups and 0% Cu-doped group (p<0.05).Abbreviations: ALP, alkaline phosphatase; BMSCs, bone marrow stromal cells; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.
Scaffolds implanted in vivo
After obtaining X-ray radiographs of distal part of femurs in which the scaffolds implanted, it was found that there was a large bone cavity for blank group and little osteosclerosis formation around the margin in the radiographs of 4-week post-implantation (Figure 8A), while closely connection between the scaffold and the new bone formation around the scaffold in 1% Cu-doped group (Figure 8D). At 8 weeks after implantation, there were a reduction of the bone cavity and osteosclerosis formation for the blank group (Figure 8B), while degradation of the composite scaffold and new bone formation occupying the cavity for the 1% Cu-doped group were observed (Figure 8E). At 12 weeks post-implantation, more osteosclerosis formation and visible boundary of the osteosclerosis for blank group were observed (Figure 8C), while the more markedly degradative scaffold was wrapped by newly regenerated bone completely and no clear boundary was found between bone and scaffold (Figure 8F). The X-ray scanning of bone after implantation demonstrated the ability of degradation and osteogenesis of the composite scaffolds.
Figure 8 X-ray images revealing the repair of bone defects in blank group and 1% Cu-doped n-CDHA/MAC group at postoperative 4, 8, and 12 weeks: (A, B and C) blank group; (D, E and F) 1% Cu-doped n-CDHA/MAC group. (A and D), 4 weeks; (B and E), 8 weeks; (C and F), 12 weeks. The red arrow represents new bone formation in bone defect.Abbreviations: n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.
H&E staining of 0% and 1% Cu-doped scaffolds implantation in the bone were observed. At 4 weeks after operation, there were some inflammatory cells in the macropores of the composite scaffolds and a few new bone formation. Also, it could be found that more blood vessel formation in 1% Cu-doped scaffolds (Figure 9A and D). At 8 weeks after surgery, inflammatory cells disappeared. more newly regenerated bone tissues were stored in the macropores of scaffolds and active osteoblasts emerged. The amount of bone matrix formed in macropores increased markedly. Blood vessels were found in 0% Cu-doped group, while there was more visible increase of blood vessels in 1% Cu-doped group (Figure 9B and E). At 12 weeks after surgery, the macropores of composite scaffolds were almost completely encapsulated by newly formed bone. And osteoblast-like cells could be seen around the bone (Figure 9C and F). At three different time points, it was found that more new blood vessels formation and bone regeneration in the macropores of 1% Cu-doped group compared to 0% Cu-doped group.
Figure 9 HE staining of n-CDHA/MAC composite scaffolds (A, B and C) and 1% Cu-doped n-CDHA/MAC composite scaffolds (D, E and F) harvested at postoperative 4, 8, and 12 weeks (magnification 40×). (A and D), 4 weeks; (B and E), 8 weeks; (C and F), 12 weeks. The red arrow represents inflammatory cells. The blue arrow represents blood vessel. And yellow arrow represents bone matrix in macropores.Abbreviations: HE, hematoxylin and eosin; n-CDHA/MAC, nano calcium-defcient hydroxyapatite/multi(amino acid) copolymer.
ONFH is a potentially devastating disease characterized by physiopathologic mechanism of circulation interruption and progressive osteonecrosis. Currently, there are rare effective methods to cure the early-stage ONFH and delay the time performing THA. According to Tan's research,27 ischemia of the femoral head and decreased differentiation of BMSC are the significant reasons resulting in the occurrence and development of ONFH. However, all of the present methods can't effectively repair ONFH and avoid the collapse of joint surface. So, based on the characteristics of ONFH, Promising composite scaffolds must be equipped with eligible biocompatibility, Three-dimensional porous structure and appropriate mechanical strength to enhance bone ingrowth and provide structural support.28,29 Besides, studies have reported doping Cu into biomaterials can enhance their original angiogenesis and osteogenesis properties.20 So, we combined the osteogenesis of n-CDHA/MAC and angiogenesis of Cu to synthesize the novel composite scaffolds. In this study, the results preliminarily revealed the biosecurity of Cu-doped n-CDHA/MAC composite scaffolds and the optimal concentration of Cu to promote angiogenesis and osteogenesis.
Studies have shown that the composite scaffold with macropore size at least 100 um and porosity exceeding 70% facilitated cell ingrowth and mineralized bone regeneration.22 And the compressive strength of cancellous bone of human being was 2–25 MPa. Our results indicated that the macropores shows uniform diameters, which were similar to the fundamental requirements. Moreover, doping n-CDHA/MAC with different mass ratio of Cu had little influence on the total porosity and compressive strength. Obviously, our prepared composite scaffolds were equipped with porous structure (approximate 81%) and suitable mechanical strength (approximate 12 MPa), which can be used for defect repair.
Appropriate degradation rate of scaffold and nontoxic degradation product are important for outstanding scaffolds. And it is commonly believed that bioactive scaffolds placed in vivo with a suitable degradation rate can provide the space for matrix deposition and bone ingrowth until osteogenesis was finished.30 In our study, the overall degradation of the scaffold (weight loss) reached almost 63% at the end of the test. And the porous structure of the scaffold has a larger surface area in contact with the immersion solution at the initial immersion, so the degradation rate is faster. During the degradation process, the amorphous part of the scaffolds degraded earlier than the crystalline part, and the degradation rate was faster. This is another factor in the early degradation of scaffolds. Furthermore, the degradation products include hydroxyapatite, different amino acids and calcium sulfate, which are not only nontoxic for the living body, but also can function as the materials of bone formation.
Cu can enhance angiogenesis and osteogenesis properties of the scaffolds via slow-release from the biomaterials with the advantage of low cost, high stability and potentially greater safety.20 The mechanism is that Cu can promote the expression of hypoxia-inducing factor-1(HIF-1) to stimulate the secretion of VEGF, which is identified as the critical factor having the strongest biological action in inducing vascularization.20,31 However, high concentrations of ions can lead to cytotoxicity.32 And there is no consensus on the threshold for toxic effects of Cu. From the study of Burghardt,33 they believed that the threshold for toxic effects of Cu on MSC was 0.5 mM. Bejarano34 doped Cu in silicate bioactive glasses and came to a conclusion that the cytotoxicity threshold of Cu was 3.0 ppm, while some researcher reported the effective Cu concentration enhancing vascularization was 14−57 ppm.20 And the study has demonstrated that human umbilical vein endothelial cells (HUVECs) were stimulated to proliferation, if the Cu concentration in culture medium was less than 32 ppm.35 Possibly, different experimental conditions and different kinds of cells used in vitro experiments might explain these results. Besides, the release of metal ions is determined by the initial dose doped in scaffolds and the degradation rate of the scaffolds. In our study, the release of Cu was consistent with the initial dose of Cu and degradation curve of scaffolds, indicating the initial dose of Cu and the rate of degradation determined the release rate. During the degradation process, there was no collapse of the scaffold observed, so no burst release of Cu was observed. Noteworthy, the slow and sustained release of Cu is possibly favourable for tissue engineering applications.
To investigate the cytocompatibility of the Cu-doped n-CDHA/MAC scaffolds in vitro, the rBMSCs were used to cultivate with scaffolds. The cell adhesion was observed by SEM and the proliferation of cells was determined with CCK assay at different time points. As described in literature,36 Adhesion is the first step of cell-material interaction, which play a key role in cell functions including proliferation and differentiation. Our results indicated that 1% Cu-doped n-CDHA/MAC composite scaffold showed better cell adhesion compared to other groups. Also, further analyses of the survival of rBMSCs were done by CCK to determine the proliferation cells cultivated with scaffolds. It concluded that obviously positive effects on cell proliferation were observed with the concentration of Cu ranging from 0 to 1%, while more than 2% showed inhibited effects on cell proliferation compared to control group. Based on these results, we considered that the optimal concentration to promote rBMSCs proliferation was 1% of the scaffolds in mass ratio and the threshold for toxic effects of Cu on rBMSCs was 2%.
Cu is a vital component of many proteins such as cytochrome oxidase and superoxide dismutase, which act as essential roles in cell activity,35 But excess Cu is toxic. The cytotoxicity of Cu was identified as the production of free hydroxyl radicals, which could participate in the disruption of membranes and organelles by inactivating essential enzymes.37 On the other hand, proper supplementary of Cu facilitates proliferation of cells on composite scaffold. Furthermore, Ewald et al has demonstrated that Cu enhances cell activity and proliferation of osteoblastic cells on brushite scaffold.38 And they also indicated that Cu promoted the expression of several proteins for bone formation including bone sialoprotein (BSP), which could stimulates adhesion, migration and survival of cells in vitro. Therefore, proper content of Cu promote cell adhesion and proliferation.
ALP, as a marker of osteogenic differentiation of stem cells, was secreted by osteoblasts.39 So, ALP expression can reveal the osteogenic differentiation of rBMSCs. In our study, higher level of ALP expression in 0.5% and 1% groups were found than in 0%, 2% and 5% groups at 7 days, indicating that the cell differentiation was quicker after being cultured on the composite with Cu ranging from 0% to 1% than other groups. Moreover, it was also demonstrated that a negative cell differentiation on scaffolds with Cu ranging from 2% to 5% compare to 0% group, showing the inhibition effect on cell differentiation when Cu content was more than 2%. Accordingly, the n-CDHA/MAC scaffolds with contents of Cu ranging from 0% to 1% showed superior ALP activity compared to that with no Cu.
Similarly, superabundant dose of Cu influences BMSCs differentiation due to the disturbance of cell activity like what mentioned above. Many scholars has reported that Cu-doped composites could enhance the bone-related gene expressions such as ALP, osteopontin (OPN) and Runx2.33,38,40,41 However, few studies illustrated the relevant mechanism of Cu stimulating osteogenesis. It was demonstrated that Cu could promote the expression of HIF-1 and initiate the transcription of hypoxia-related genes such as VEGF. Then, activation of VEGF receptors resulted in the expression of genes including Runx2, Bone Morphogenetic Protein (BMP) and OPN, which are stimulating factors of osteogenesis.42,43 This might account for stimulation of cell differentiation.
The in vitro experiments proved that 1% Cu-doped n-CDHA/MAC promoted the proliferation and differentiation of rBMSCs, indicating a good potential to induce bone formation. We used an in vivo model to investigate the degradation property, as well as the osteogenic and angiogenic capability. The scaffolds with a suitable degradation rate placed in vivo can provide the space for matrix deposition and tissue ingrowth until healing was finished.30 The X-ray imaging of 1% Cu-doped group at 4, 8, 12 weeks showed the capacity of degradation and osteogenesis compared to blank group, which was similar to the results of the experiments in vitro. H&E staining of 1% Cu-doped group showed osteoblast-like cells beading at the interface between bone and scaffolds, obvious new bone formation and bone ingrowth into macropores of the scaffolds, together with more blood vessels formation compared to 0% Cu-doped group, indicating that Cu in the scaffolds resulted in superior osteogenesis and angiogenesis.16–18,20,33 Because deficiencies in oxygen and nutrient exchange at the necrotic area are a major issue due to lack of vascularization. So, adequate blood supply is the prerequisite for formation of bone and osteogenesis will vary according to the vascularization of the site.44 Highly expressed factors mentioned above synergistically recruit stem cells and endothelial cells into bone defects to enhance vascularization and promote osteogenesis. Thus biomaterials with proper contents of Cu showed better osteogenesis and angiogenesis in vivo than that with no Cu. And our study came to the analogous conclusion: Cu-doped n-CDHA/MAC composite scaffolds with the contents of Cu 1% exhibited outstanding angiogenesis and osteogenesis.
This study has preliminarily demonstrated Cu-doped n-CDHA/MAC was equipped with properities of osteogenesis and angiogenesis. And we will future investigate the mechanism of osteogenesis and angiogenesis of the composite scaffolds.
In this study, Cu-doped n-CDHA/MAC composite scaffolds were prepared. Cu-doped n-CDHA/MAC scaffolds were degradable and showed satisfactory mechanical property (approximately 12 MPa). Also, the Cu-doped scaffolds presented slow and sustained release of Cu and exhibited remarkable influence on the osteogenesis and angiogenesis. Compared to no Cu-doped scaffolds, 0.5% and 1% Cu-doped ones showed stimulation, while 2% and 5% were demonstrated inhibitation on bone regeneration. And prepared 1% Cu-doped n-CDHA/MAC scaffolds combined characteristics with optimal osteogenesis capacity. It was also found that the scaffolds doped Cu ranging from 0% to 1% in mass ratio will promote the proliferation and differentiation of rBMSCs. So, doping n-CDHA/MAC with 1% Cu is safe and can provide a promising approach for enhancing the capacity to heal of bone defect. This new biomedical scaffold with the properities osteogenesis and angiogenesis might be used to treat ONFH in the future.
This research was funded by the National Natural Science Foundation of China Program (81501873), Science and Technology Project of Sichuan Province (2015SZ0085). Study approval was obtained from the Biomedical Ethics Committee of West China Hospital, Sichuan University (2017046A). We followed laboratory animal guideline for ethical review of animal welfare for the animal according to National Standard of China during the whole animal experiments. P.M. and H.P. contributed equally to this work and should be considered as equal first authors. The data supporting the results reported in the manuscript will not be shared due to our further research on the composite scaffolds.
The authors report no conflicts of interests in this work.
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Ep. 21 | Longing, Ripening and Dada Vaswani
By Sarina Rao September 24, 2019 All Podcasts, Blog, Call and Response Podcast, Foundation Updates, Offerings
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{Photo by Krishna Das, Auckland Harbor July 2018}
Ep. 21 Longing, Ripening and Dada Vaswani
The other people who met Maharajji talk about is the experience of His unconditional love, as you said once, "His unbearable love," and my guess is that for many people, certainly who've not had the experience of Baba in the body and who maybe don't have… my guess is when you speak of that, many people wish to feel that, to feel unconditional love, maybe that's why they keep coming, I don't know. And so, after a lifetime of doing this and becoming a teacher, what can you say? You know, what can you say to people about having that experience, finding that experience?
"I would say that the more you long for it, the better it is. Period. You have to be where you are. You can't be anybody else. You can't be anywhere else. What you can do is look, be yourself 100% and the longing for that love is what brings towards it. Period. End of conversation. That's it. Nothing else. That's my only qualification for any of this, is longing. This insane amount of longing that destroyed my life. It destroyed everything I thought I was going to be in life and replaced it with all this nonsense. It was that longing which I brought into this life and when I speak about this love and my Guru and you feel "I want a guru, I want a guru," that's a good longing to have because that longing will not leave you until you find what it is you're longing for. There's no easy way to get through this. It sucks. Period. I can't make it all right for anybody but I can tell you that the more longing you have, the better it is. " – Krishna Das
Q: Hi. Ram Ram.
KD: Ram Ram.
Q: I'm Annapurna.
KD: Annapurna. Annapurna.
Q: The gentleman who asked about finding, looking for a guru, finding a guru, one of the more compelling things that you and the other MTM's talk about…
KD: Use words, ok?
Q: the other people who met Maharajji talk about is the experience of His unconditional love as you said once, His unbearable love… and my guess is that for many people, certainly who've not had the experience of Baba in the body and who maybe don't have… my guess is when you speak of that, many people wish to feel that, to feel unconditional love, maybe that's why they keep coming, I don't know. And so after a lifetime of doing this and becoming a teacher, what can you say…
KD: I'm not a teacher, thank you.
Q: You know, what can you say to people about having that experience, finding that experience…
KD: I would say that the more you long for it, the better it is. Period. You have to be where you are. You can't be anybody else. You can't be anywhere else. What you can do is look, be yourself 100% and the longing for that love is what brings towards it. Period. End of conversation. That's it. Nothing else. That's my only qualification for any of this, is longing. This insane amount of longing that destroyed my life. It destroyed everything I thought I was going to be in life and replaced it with all this nonsense. It was that longing which I brought into this life and when I speak about this love and my Guru and you feel "I want a guru, I want a guru," that's a good longing to have because that longing will not leave you until you find what it is you're longing for. There's no easy way to get through this. It sucks. Period.
I can't make it all right for anybody but I can tell you that the more longing you have, the better it is. So what, I shouldn't talk about Maharajji because it makes people unhappy? That unhappiness is a good thing. And that's what happened when I met Ram Das. This longing was the longing I already had for something that I didn't even know what it was, it got crystallized in a way. It got concentrated. It got super infused and then nothing kept me away from that. And it just worked out the way it worked out. You know, many of you probably know the story but, so, after about a year and a half of hanging out with Ram Das and I recognized that it was Maharajji that was coming through Him, I said, you know, "I want to go see Maharajji" and he said, "Well, I can't tell you where He is." He couldn't even, He didn't even tell us his name. Because when he left India, when Maharajji, when he was going home, Maharajji said to him, "Don't talk about me in America." So of course, all he did was talk about him, but he never said his name. And he never said where you could find Him. But he talked about Him. So I said to Ram Das, I'm going to India. And he said, "Well, I can't tell you where to go." I said, "I don't care, I'm going. I'll find Him." And he said, "Ok, ok wait. I'll give you my friend's address in Nainital," which is this little town up in the hills, "and you can write to him and see what happens." So his friend's name was KK. KK Shah. Now, KK had grown up in Maharajji's lap from the year, 8 years old, really literally in his lap. Maharajji was like his grandfather and they had the most intimate, sweetest relationship and KK was, also had the personality of a spoiled child. If Maharajji said, "Sit down." He would stand up. If Maharajji said "Stand up," He would sit down. Go away, he would stay. Stay, he would go away. Eat, he would fast. Fast, he would eat. That's the way he was, you know? So, after Ram Das met Maharajji the first time, that day KK was there. He was his translator and Maharajji said to KK, "Take him home and take care of him and serve him and feed him and make sure he has what he needs." So, Maharajji had given KK this seva or this service to do for Ram Das. So now, I wrote to KK and I said, you know, I would like to come and see Maharajji. So he wrote back to say, "You know, He's not in the hills at this time, but when he returns I'll write back to you and I'll let you know."
So about a month later, or a month or two later I got a letter back saying, "Maharajji," he still didn't say his name, right? "Maharajji has returned to his temple in Kainchi." Aha, I have a place. "And after some days I was able to see Him. As you know, Great Saints do not encourage the devotees to come to them. But their doors are always open." Great! And then the next line was, "And if you're traveling in India, you can come see Him." Fantastic! Here we go, right? Not exactly.
Many years later, KK says to me, "Did I ever tell you what Maharajji said that day?" I said, "No." He said, "Well, I came to the temple with, now there were three letters, my letter and two other guys, I came to the temple and I came into the room where Maharajji was and I put the letters on this bed next to Him and I sat down on the floor and I began peeling an apple and cutting it into small pieces to feed it to Maharajji." He used to buy these very soft apples because he had three teeth, so he'd cut them small. So he was cutting the apple. He was feeding Maharajji the apple and Maharajji was talking to these other people in the room and He looks down and He sees the letters and He says, "What's that?" And KK said, "They're letters from students of Ram Dass. They want to come see you." "Nay. Tell them not to come. What do I have to do with this?" And He goes back to His conversation.
Now, see the play right? KK got very upset and he began to pout because Maharajji had given him the job of helping Ram Das, so to speak. The seva. And now Maharajji was interfering with the very work that He gave KK to do and this was unacceptable. So he began to pout and he stopped feeding Maharajji the apple. And Maharajji, and he looked down like this. Maharajji said, " KK what's the matter?" And he, KK showed me, he tried to push his head up like this. KK would look away. Then he'd let go and he'd put his head back down like this. Finally, Maharajji said, "Ok. Say what you want. Tell them what you want." So, what did he write? Ok? What did he write?
He's not going to lie. He's a good devotee. But he wrote, "As you know, great saints like Maharajji do not encourage the devotees to come to them but their doors are always open. So if you're traveling in India…" My whole life hanging on a thread. Oops.
Or so it seemed, right?
But I, great… all I thought was "I'm going." I was going to go, I didn't care what happened. So, I had that longing, that pushed me. Everybody has that but we kill it mostly because we don't believe we can ever really be happy. We don't believe we're worthy of love. We don't believe we can be loved and we can love. Fully. And so we kill that longing. We try to numb it, numb ourselves down with everything we do. That's why the stronger the longing is, the better it is. You know that Rumi poem, you must have heard "Love Dogs"? I'll screw it up, but you know, it's like "A Man is Crying 'Allah Allah' and his lips grew sweet with the praising… and this cynic walks by and says, ' why are you calling out, have you ever got a response?' and the guy thinks "he's right and he quits singing.' And he goes to sleep and he has this dream where the guide of souls comes to him and says, "Why did you stop calling out?" And he said, "Because I never got a response." And he said, " The calling out is the response." You see. He said, "You hear that dog moaning in the distance, calling out for its master? That's the connection."
We think we're doing it. We think it's up to us. We think when we call out that's the first thing and we're waiting for a response. The calling out is the response of the longing that comes from our own soul, our own Being. That's the chanting. So, when you say the Name right once, which means you're fully there, completely immersed in the longing, in the presence, then of course the real Ram will come.
It's not like it's not here already but we don't see it.
That longing, it saves our, it's the only thing that can save us and on the other hand, it fucks up our whole life completely because nothing will ever be enough, no matter how much we try to fake it, we don't know we're faking it. And we're good at faking it.
That's ok. Go ahead, fake it.
Maharajji said, keep lying. Keep singing the false Name. Don't stop. One of these days, it'll come out of you the right way. You have to keep calling out. If you don't do it, what will happen? Nothing. And of course, the Name, chanting the Name is only one of the many practices and anything can be a practice if it's done the right way, if it's done with awareness, with intention. The trick is to be able to give yourselves and give ourselves 100% to it. And we don't do that. I remember the winter of '69-'70, Ram Das and I and others were up at the Lama Foundation all winter, and we heard about this New York artist named Herman who had gone to India and learned to meditate and had come back and was now living just down the mountain from the Lama and so a group of us went to see him and I sat in the back of the room and everybody else was talking. I was just listening and stuff and then we were leaving a couple of hours later and I was the last one out the door. He grabbed my arm and he looked at me and he said, "You, you have to find out why it is that you can't give yourself 100% to whatever you're doing." You ever see, like, in a taxidermy shop, you know? The squirrel on the wall? That was me. He nailed me to the fucking wall. It was unbelievable. I can remember that feeling right now. That was 50 years ago. He got me perfectly and from that moment to this moment, I notice that I'm not giving myself 100% to it so then I recommit again and again. I come back. It's… so at one point, I would assume that I'll come back and I'll never leave. We'll see. It's a nice fantasy.
Q: Ah Krishna Das, I want to say thank you so much for coming out to LA. Our mutual sister, who we were going to actually fly to Atlanta with, Geeta…
KD: Oh yeah, Geeta. Lovely. We had a great time.
Q: To come and see you , so thank you so much.
KD: Are you a disciple of Dada?
Q: Exactly. Dada talks about, when you talk about ripening the soul, He talks about the ego and once we can start to diminish that I think that's where the ripening process begins. So before you answer that, I just have a request, either today or tomorrow, if you can sing "God is Real" because I have watch it about 50,000 times and I just can not get…
KD: So you know it by now. Give it up.
Q: I want to hear it from you. I want to hear it from you.
KD: This is the weirdest thing. I want to tell you, for some crazy reason, every place I've been going the last month or so, people have been asking me to sing "God is Real." After like, years. I hardly ever sang it. Everybody wants… what's the deal here?
Q: I think I've been sharing it all over social media so that's why.
KD: Terrific. So, Dada Vaswani, who left His body last year at 100 years old was one of the greatest saints I've ever met. This man, there was nothing in there but love. It was extraordinary. And he was so kind to me. The first time I met him was in Chicago and this guy Jeremy who made the movie of me, you know? Whatever it's called. One Track Heart. Yeah. He was in Chicago shooting and he knew the people where Dada was staying. So we went over there. And so I sang for Dada and he just sat there. He was in a wheel chair at the time. He was 96 maybe at that point or 95. And after I was finished singing, this Indian guy goes "Oh Dada, when Krishna Das sings it makes me dance…" and Dada kind of opened up his eyes. He looked at the guy and said, "When Krishna Das sings, it brings my mind to peace." Very nice. I thought, that was very nice. Thank you.
But so then last year, a year and a half ago, he would come to New York and he would ask me to come and sing at his talks and he would always, he would talk first and then he would sit on the side of the stage and listen while we sang. So the last time I saw him after we chanted, we went over, I went over there to see him and I was sitting on the ground in front of him, and he just looked at me and he said to one of his people, he said, "Go bring those books for Krishna Das." And she said, "Dada, they're all the way on the other side of the room in the hallway" "Yeah, go get them go get them." So this woman goes off to get the books for me. And he just sat there and he was looking at me and smiling, just looking and smiling, like 15 minutes went by, right? And another one of his devotees said, "Dada, Krishna Das is sitting here but you're not talking." And he looked at her and then he looked back at me and he smiled again and he said, "Ralph Waldo Emerson was a very great yogi and he wanted to meet this other guy in England," I forget who it was, "and he took a horse carriage and then he took a train and then he took the boat and then he goat to England and he took another train and then a carriage and then he walked and he got to this other guy's, this great saint's place in England, this yogi's place in England," and this was in the 1800s, "and they went in and they sat for an hour together without a word being spoken and then Ralph Waldo Emerson left and walked and took the train and took the boat and went back home." And then he looks at her and he looks over at me and he says, "Krishna Das and I have said everything there is to say." That was the last time I saw him.
He was so beautiful. God.
That there are Beings like that, it's inconceivable. We slog around in our miserable lives and there are Beings like that in the world. And we are like that. But we don't see that. It's extraordinary. Kindness. Kindness.
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Swami Vimalananda, My Beloved Elder
Ep. 20 | Compassion for Abusers
Ep. 19 | Spiritual Love and Worldly Love
Ep. 18 | How To Develop Faith
Celebration of Life Remembering Ram Dass – Free Event
Monday, Feb 10, 2020 @ 3:00 pm - 5:00 pm HST
Kahului HI
Path of Devotion Retreat with Krishna Das & Friends
Friday, Feb 14, 2020 - Sunday, Feb 16, 2020
Garrison NY
Garrison Institute Friday Kirtan
Friday, Feb 14, 2020 @ 8:00 pm - 10:30 pm EST
KD's new album is here!
Peace of my Heart
Click here to get your copy!
"The way I sing, I try to let the mantra do the work. I simply pay attention to the prayer and repeat it with as much of my being as I can. I don't try to manipulate emotions, I don't try to manufacture any kind of feeling at all, it naturally arises." ~ KD

redpajama_set_name: RedPajamaCommonCrawl


Jocelyn Zichterman is an abuse advocate and whistleblower fighting against the IFB Church. Zichterman was a critical source and interview for the ABC News 20/20 investigative hour Shattered Faith as well as the "Ungodly Discipline" series featured on CNN's Anderson Cooper 360. She has also been a source for The New York Times and The Huffington Post, among many other publications. Jocelyn lives in Oregon with her husband and eight children.


These did not fit the person I got them for, so they're up for sale for a good deal. I believe they're factory seconds, but they're really pretty nice. Please see photos for any wear, damage, or scuffing. Whatever there is, I made sure the photos highlight it and make it look much worse than what one would actually notice when the shoes are worn.
They were worn only a few times outside. Still in near new condition as far as the sole and functionality is concerned.


text:
Most beautiful locations where TV shows were filmed
By Rachel Monday - December 10, 2018
How often have you wished you were a part of your favorite TV show? While landing a role on the series might not be easy, there is a way you can get involved without busting out your acting chops. Although a lot of these shows are filmed on sets, some of their scenes are based in real-world locations that are easy to visit. Time to break out the passport and get ready for a vacation.
Dark Hedges (Game of Thrones)
It's amazing that anyone's still alive in Game of Thrones. With the way the body count racks up in that show, we thought they'd have all taken each other out by now. Nevertheless, there are still some characters left, and they continue to be popular with millions of fans around the world. Luckily, even though the show itself is so dark and destructive, the places where it's filmed are actually surprisingly beautiful. Take Dark Hedges in Northern Ireland, for instance. It might be known as the terrifying Kingsroad in Game of Thrones, but in reality, it's a nice place for a serene, scenic stroll. Don't believe us? Check it out for yourself.
Pasadena City Hall (Parks and Recreation)
We're not that used to seeing the outside of Pasadena City Hall on Parks and Recreation, but the few glimpses we get are definitely worth it. Despite being a government building, it's beautifully crafted and a real wonder to see up close. In the series, it famously acts as Pawnee City Hall where Leslie Knope and the rest of her eccentric colleagues regularly spend their time. While the interior might have been a set, the outside was 100% authentic.
Highclere Castle (Downton Abbey)
Downton Abbey might have appealed to the hardcore historians and period drama lovers, but they were just a fraction of the show's overall audience. Millions fell in love with the aristocratic Crawley family, and not just because their drama was so enticing. Did you see where they lived? They had some sweet digs on Downton Abbey, and now you can see them for yourself. Although only certain parts of it are open to the public, you can visit Highclere Castle in Hampshire if you want to feel like one of the Crawleys. Given how gorgeous the grounds are, it's worth the visit.
Police Beach (Lost)
Few shows have gotten viewers as hooked as Lost did back in the 2000s. Despite what you may have thought of the finale, people couldn't get enough of the show when it was still on TV. Critics today still label it as one of the best series ever made, and we get why. It knew what it wanted to be, and it pulled it off expertly. If you were a massive fan of the show, why not visit the beach where most of it was set. Luckily, you don't have to crash land to get to it. It's in a part of Hawaii known as Oahu, with the beach itself aptly named Police Beach. Coincidence? Apparently so.
Seen somewhere that's caught your eye? Now's your chance to become the star of the show!
How to avoid long customs and immigration lines on your next trip
Emily Alex
So you've just sat on a plane for thirteen hours traveling from Indonesia, what you'll then have to ...
How to see the world without leaving a trace behind you
Mia Pazzo
Millions of people travel the world each year, moving from country to country; whether it's going ...
Assaf Bitton
The Colossus of Rhodes was one of the greatest Wonders of the Ancient World, as well as an enduring ...
Our goal is to faithfully supply our readers with engaging, entertaining, and informative stories that are related to the exploration and discovery of all things travel oriented.
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Report shows more people taking advantage of traveling in an RV
Lali Odena January 12, 2021
Best vegan-friendly places in the world
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2021 'travel boom' among Americans less likely than thought
Moran Sapir January 7, 2021
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What It's Like Going on a Safari Trip Amid the Coronavirus Pandemic
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text:
Information on Coronavirus
Department of School Counseling & Related Services
Emergency Food Service Plan
Click Here for the Food Services Department
January 3rd, 2022 Update:
Dear School Community,
Grab and Go style breakfast and lunch will be served at every school site with the exception of the Dual Language School in Fords. Meals will be available between the hours of 10:30am to 1:30pm Monday - Friday. Students and families can come to the school closest to their home and pick up a quality meal during this time. All COVID protocols must be strictly adhered to when picking up meals. If you are unable to pick up a meal, and require one please contact your principal for further assistance.
August 31, 2021 Update:
In accordance with the updated meal distribution guidelines, summer meal distribution will come to an end on Tuesday, August 31st at all meal distribution sites. Meal service will resume on Tuesday, September 7th, 2021 at all school sites for all students during the course of the school day. It has been our pleasure to serve you and we look forward to the start of the new school year. Thank you.
June 25, 2021 Update:
We are happy to inform you of our continuous, uninterrupted meal service. Meal distribution will take place on Monday, June 28th, Wednesday, June 30th and Friday, July 2nd from 10:30-1:00 pm at the following sites:
Perth Amboy High School- Main Campus
Rose M Lopez
Robert N Wilentz School
There will be no meal distribution on July 5th in observance of July 4th holiday. Beginning on July 6th, meal distribution will take place at any school hosting a summer program and at sites hosted by the Perth Amboy Recreation Center. It is always our pleasure to serve you and look forward to seeing you once again!
June 9, 2021 Update:
Meal service and distribution will resume to a regular schedule on Wednesday, June 9th 2021 from 10:30 am to 1:30 pm at all district schools and at the Perth Amboy Office of Recreation from 12:30pm to 4:00pm. As a reminder, students and parents have the option of going to any of the school sites closest to their home to pick up meals daily and no ID cards are required at this time.
It is always our pleasure to serve you and look forward to seeing you once again!
In observance of Election Day, the Perth Amboy School District will be closed tomorrow, Tuesday, June 8th, 2021. Regular meal distribution will take place today, Monday, June 7th 2021 where parents/students can pick up meals for 2 days. Meal distribution will resume on Wednesday, June 9th to a regular schedule at all schools and at the Perth Amboy Office of Recreation from 12:30pm to 4:00pm.
Meal service and distribution will resume to a regular schedule on Wednesday, June 2nd, 2021 from 10:30 am – 1:30 pm at all district schools and at the Perth Amboy Office of Recreation from 12:30pm to 4:00pm. As a reminder, students and parents have the option of going to any of the school sites closest to their home to pick up meals daily and no ID cards are required at this time.
May 27, 2021 Update:
In observance of the Memorial Day Holiday, the Perth Amboy School District will be closed on Monday, May 31st, 2021 and Tuesday, June 1st, 2021. Regular meal distribution will take place on Friday, May 28th, 2021 where parents/students can pick up 3 meals. Meal distribution will resume on Wednesday, June 2nd to a regular schedule at all schools and at the Perth Amboy Office of Recreation from 12:30pm to 4:00pm.
April 11, 2021 Update:
Meal service and distribution will resume to a regular Monday, Wednesday, Friday schedule on Monday, April 12th, 2021 from 10:30 am – 1:30 pm at all district schools and with the exception of the Dual Language School in Fords, Perth Amboy Adult School and the 9th Grade East Campus. Meals are also available for pick up at the Perth Amboy Office of Recreation from 12:30pm to 4:00pm.
As a reminder, students and parents have the option of going to any of the school sites closest to their home to pick up meals daily and no ID cards are required at this time.
April 1, 2021 Update:
In observance of Spring Break, the Perth Amboy School District will be closed from April 2nd 2021 through April 9th 2021. Meal distribution will continue to take place on the following days: Monday April 5th, Wednesday, April 7th and Friday, April 9th at the following sites:
Perth Amboy High School Main Campus
300 Eagle Avenue – 10:30 a.m. to 1:30 p.m.
The Perth Amboy Office of Recreation
1 Olive Street – 12:30 p.m. to 4:00 p.m.
In observance of the Spring Break, the Perth Amboy School District will be closed starting on Friday, April 2, 2021. Meal distribution will take place on Monday, March 29th, 2021, Wednesday, March 31st and Thursday, April 1st.
During Spring Break, meal distribution will continue to take place on the following day: Monday, April 5, Wednesday, April 7th and Friday, April 9th at the following sites:
February 15, 2021 Update:
In observance of the Presidents Day Holiday, the Perth Amboy School District was closed today, Monday, February 15, 2021. Meals will be distributed on Tuesday, February 16th, from 10:30am to 1:30pm, Wednesday, February 17th and Friday, February 19th, at all schools with the exception of the Dual Language School in Fords, Perth Amboy Adult School and the 9th Grade East Campus. Meals are also available for pick up at the Perth Amboy Office of Recreation from 12:30pm to 4:00pm.
In observance of the Presidents Day Holiday, the Perth Amboy School District will be closed on Monday, February 15, 2021. Regular meal distribution will take place on Friday, February 12th where parents and students may receive meals for 3 days. Meals will be distributed on Tuesday, February 16th, from 10:30am to 1:30pm, at all schools with the exception of the Dual Language School in Fords, Perth Amboy Adult School, and the 9th Grade East Campus. Meals are also available for pick up at the Perth Amboy Office of Recreation from 12:30pm to 4:00pm.
January 21, 2021 Update:
Due to the increase in COVID 19 cases in the community starting on Monday, January 25th we will modify the meal distribution days to Monday, Wednesday and Friday. We will continue to serve meals from 10:30am-1:30pm at all distribution schools with the exception of DLS, East Campus and the Adult School and at the Perth Amboy Office of Recreation, located at 1 Olive Street, from 12:30pm to 4:00pm. Each parent/student will receive 2 meals at pick. Thank you.
In observance of the Dr. Martin Luther King Jr. Holiday, the Perth Amboy School District will be closed on January 18, 2021. However, meal distribution will continue to take place on Monday, January 18, 2021 at the following sites:
December 21, 2020 Update:
The Perth Amboy School District will be closed from December 24th through January3rd. Regular meal distribution will take place on December 22nd. In addition, we will provide Breakfast & Lunch on December 23rd, December 28th and December 30th, for multiple days, at the following sites:
November 25, 2020 Update:
Although the Perth Amboy School District will be closed from November 26th and 27th for the Thanksgiving holiday, we will be providing Breakfast & Lunch, on November 27, 2020 from 10:30 a.m. to 1:30 p.m. at the following sites:
October 30, 2020 Update:
Although the Perth Amboy School District will be closed from November 2nd through November 6th we will continue to provide Breakfast & Lunch, on Monday, November 2, 2020, Wednesday, November 4, 2020 & Friday, November 6, 2020 at the following 4 sites:
Robert N. Wilentz School
51 First Street – 10:30 a.m. to 1:30 p.m.
Rose M. Lopez School
435 Seaman Avenue – 10:30 a.m. to 1:30 p.m.
Perth Amboy Recreation Center
July 2, 2020 Update:
Dear Perth Amboy Community,
We are happy to inform you of our continuous, uninterrupted meal service. We are also serving tomorrow July 3rd and we will continue to serve throughout our summer break from our same 4 sites, during the same hours, regardless of school closures. Additionally, the following 5 sites throughout the city will also have meals available for pickup starting Monday July 6th, and throughout the summer break.
8:00 am at Anthony V. Ceres Elementary School located on 445 State Street
11:00am at PATTEN PARK located on 338 Reade Street
11:00am at FRANCIS ST PARK located on 972 Francis Street
11:00am at HARBOR TOWN PARK located on 1 Barnes Ct
11:00am at WASHINGTON PARK located on 660 New Brunswick Ave
Click Here for May 5, 2020 Reminder
The district is committed to serving our students during this difficult time. Breakfast, lunch, and a modified dinner will be served to ALL Perth Amboy children.
A grab-n-go bag that will contain a pre-packaged breakfast, lunch and a sandwich for dinner will be made available. Children will be able to pick up these meals at the main entrance of the following locations:
When: Monday, Wednesday and Friday
Time: Between the hours of 8:00 AM – 11:00 AM
Locations:
Perth Amboy High School - 300 Eagle Avenue
Rose M. Lopez School - 435 Seaman Avenue
Robert N. Wilentz School - 51 First Street
An additional site has been added
Perth Amboy Office of Recreation - 1 Olive Street
Breakfast Items:
Fruit Loops
Apple Jacks
Lunch & Dinner Items:
Ham & Cheese Croissant Sandwiches
Lunchables which consist of crackers, ham & cheese
Lunchables which consist of pizza
Turkey Ham & Cheese Sandwiches
Turkey Breast Sandwiches
Turkey Combo Sandwiches (Ham, Bologna, Salami)
Italian Turkey Combo Sandwiches (Ham, Salami, Pepperoni)
Grilled Cheese Sandwiches (Prepackaged)
Variety of Fresh Fruit & Vegetables: Apples, Apple Slices, Oranges, Carrot Sticks, & Celery Sticks
Milk Option: Fat Free White, 1% White, Chocolate, & Strawberry Milk
Please click the link below for more detailed information.
PAPS Emergency Closure Food Service Plan
MyCentralJersey.com News Article
NJ.com News Article


text:
Braqq braqq.com
Home/bulgaria/Borisov welcomed the residency of "Euxinograd" Vucic, Tsipras and Dunkin
Borisov welcomed the residency of "Euxinograd" Vucic, Tsipras and Dunkin
bulgaria November 2, 2018 bulgaria
Borisov Tsipras welcomes. Photos of government press
Prime Minister Boyko Borisov welcomed Prime Ministers of Greece and Romania – Alexis Tsipras and Viorica Dankila and President of Serbia Alexander Vucic, who are participating in the four-party summit of Bulgaria, Greece, Romania and Serbia, the government's press service.
The Bulgarian Prime Minister thanked his colleagues for his serious commitment to the traditional form, which he resumed in Varna in October 2017. According to Prime Minister Borisov, the fact that this is the second meeting of the last six months demonstrates the productivity of this format, which contributes to deepening regional cooperation and developing good neighborly relations.
Four of our countries are jointly responsible for strengthening the security, economic development and interconnection of the region, the Bulgarian Prime Minister stressed before his colleagues before the 4th meeting. Prime Minister Borisov expressed his confidence that negotiations on projects of common interest will continue, so that the agreements reached in an optimum manner will be achieved in order to ensure the interconnection, security and stability of the South East Europe region. According to the Prime Minister, the presence of Israeli Prime Minister Benjamin Netanyahu is also in the interest of fruitful multilateral dialogue.
Subscription for printed or electronic "24 Hours" and other Media Group Bulgaria releases.
Because of theft? Maradona took millions away from her beloved daughters
In 2037 with new cases of coronavirus in our country
Prince Philip had at least 12 girlfriends in 73 years of marriage to the Queen of Great Britain
Samsung will make the new 5G chips from Qualcomm-World
Stunning beauty loves hot PHOTOS 18+
In 2037, 34% of those tested were recently infected with COVID-19, 996 were cured
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Color: black
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Hysteric Glamour

This is from my personal collection

Color: black

Size: medium

buy here

redpajama_set_name: RedPajamaGithub


Who are Good Candidates for Cheek Implants?
Cheeks are one of the things that we first notice in a person's face. Plump and elevated cheeks give out that youthful appearance, which is why some people would say that cheeks are one of the key areas of the body which determines your age.
Aging, unfortunately, makes the cheeks sag due to volume loss. Strenuous physical activities such as weightlifting may also be the reason why there's volume loss on your cheeks which results in a deflated appearance.
Cheek implants also called as malar implants are one of the more enduring and permanent surgical methods that can bring back the volume of the cheeks that lacked contour.
This type of surgical method is proven safe and pretty straightforward, and the postoperative recovery does not take a long time.
If you've tried and enjoyed the results of dermal fillers and wants to have long-lasting, enhanced and plumped cheeks, then cheek implants are the way to go.
A flat face that lacks in contour, this might be due to genetics or aging.
The structure of the cheeks is limp.
Lost cheek volumes, because of strenuous physical activities and aging.
Then you can be a good candidate for cheek implants. Be sure to consult a trusted surgeon first to know which procedure would work best for you.
Where your surgery will be performed. Is it an ambulatory, or an office-based, or a hospital facility? The cost will differ according to the location where your surgery will be performed.
Your surgeon. How well-known and the level of the expertise of your surgeon will be put into consideration.
Where your surgeon is located. A surgeon's professional fee may vary according to where he or she is located because some cities have a higher cost of living.
Medical tests and check-ups might be needed before the surgery. You may need to undergo medical tests and follow-ups with your surgeon to avoid complications during and after surgery.
Anesthesia. Will it be a local anesthetic with IV, oral sedation or a general anesthetic?
Different options will be discussed with your surgeon regarding the surgical techniques and anesthesia. A local anesthetic with IV, oral sedation, or a general anesthetic can be used when performing a cheek implant surgery.
The implants are usually inserted through the mouth, or underneath the lower eyelids. If inserted through the lower eyelids, the incision will be made beneath the eyelids, so that the eyelashes can conceal the incision.
The most popular material used is solid silicone implants. Other options include Gore-Tex® and Medor® implants. Gore-Tex® is made from expanded polytetraflouroethylene. Medor® is an implant which comes with the facial tissue to integrate the implant with the bone over time.
The duration of the cheek implant surgery alone usually takes about 30-45 minutes.
When you have decided to undergo surgery and get the results that you're hoping for, you should prepare well for the surgical procedure.
Following your surgeon's pre-operative instructions is vital to the success of your surgery.
You're required to have medical evaluations and testing before the surgery. This is to ensure your readiness for the surgery and to avoid complications during the surgical procedure.
No drinking and smoking two months before the surgery.
You should provide a detailed and accurate medical history. This should include the medications that you're currently taking.
To avoid increase bleeding during the surgery, aspirin, anti-inflammatory drugs or herbal supplements should not be taken before the surgery.
You should be accompanied by a loved one or a friend when you undergo surgery. This way, you won't have to go to the surgical facility by yourself. Having someone drive you to the facility will make you feel at ease because surgeries can make you feel anxious.
Bruising and swelling after surgery is normal. However, it should subside within two weeks. Cold compresses help in minimizing the discomfort. Make sure to apply it to the area where the incision was made.
If incisions were made in your mouth, always rinse with mouthwash to minimize the possibility of infection at the incision area.
If incisions were made beneath your eye, your eyes should be lubricated with eye drops as often as possible. It's best that you consult your post-operative surgeon which eye drops you'll have to use.
Rest and avoid any physical activities that'll require you to exert a lot of effort for six weeks. Physical activities might misplace the implants and open the sutures.
To prevent the fluid from accumulating around the implants, you should keep the head elevated, especially during sleeping time. Find yourself a comfortable sleeping position that'll enable you to raise your head.
It's best that you eat soft and nourishing food for ten days to avoid discomfort caused by chewing solid food. Having a soft diet can ensure that the sutures in your mouth won't be disturbed.
To minimize swelling and make sure that implants stay in place, you should wear the bandage provided to you for a week after the surgery.
Only return to work or your daily activities when you have received the go-signal from your surgeon.
Never miss follow-up appointments with your plastic surgeon.


One helpful component of divorce or legal separation is the parenting plan you will lay out with your ex, that serves as a guide to how you will continue to parent your children in the wake of the divorce. A parenting plan is a document written by both you and your ex, that determines how you will divide regular time, holidays, vacation time, and major parenting decisions with your ex. It is a clear outline that will help you stay on the same page as your ex, thus reducing conflict for the future and making it possible for both of you to set realistic, agreed upon expectations of how things will go.
family vacations, holidays, birthdays, etc.
It is important that you enter the process for forging such a plan with a set of predetermined goals, and think through each goal beforehand, so that when it comes time to sit down and negotiate the terms you don't end up leaving the table without securing what you want. You should take into consideration your child's needs and balance them against your own.
Very young children may struggle more with divorce. During the first three years of their life, a child is especially vulnerable; a study from Australia suggests that it is during these years that they form they ways in which they deal with stress and their emotions. It discourages dragging children back and forth for overnight stays between different homes at this time; this will more than likely undermine the stability the child feels. More important is developing a deep, nurturing relationship with both parents through plenty of time together.
It also suggests parents develop consistent rules and expectations between the two parents during visits, so they don't have to continually adapt and will feel the additional stability. Both parents should focus on providing active and engaged play time, and purposeful down time with just that parent.
Keep in mind that it is typical for families to take 2 years to fully return to normal post-divorce–perhaps you need to draw up a parenting plan to reflect this tumultuous time, or a temporary parenting plan. Then, when they are older and less vulnerable, it may make sense to change the terms of the agreement. Either way, make the plan clear–this will reduce conflict, keep both parent's emotions in check, and make it easier for your child to adapt.
If you have difficulty agreeing on plans with your ex, consider a mediator, who will ensure the process remains even and productive.


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Волинський окружний адміністративний суд — місцевий спеціалізований адміністративний суд першої інстанції, розташований у місті Луцьк, юрисдикція якого поширюється на Волинську область.

Компетенція
Місцевий адміністративний суд при здійсненні судочинства керується Кодексом адміністративного судочинства України. Він розглядає адміністративні справи, тобто публічно-правові спори, у яких хоча б однією зі сторін є органом виконавчої влади, органом місцевого самоврядування, їхня посадова чи службова особа або інший суб'єкт, який здійснює владні управлінські функції. Іншою стороною є приватний елемент (громадянин, юридична особа приватного права тощо).

До числа адміністративних справ належать, зокрема, спори фізичних чи юридичних осіб із суб'єктом владних повноважень щодо оскарження його рішень (нормативно-правових актів чи правових актів індивідуальної дії), дій чи бездіяльності; з приводу прийняття громадян на публічну службу, її проходження, звільнення з публічної служби; щодо виборчого процесу тощо. В окремих випадках адміністративний суд розглядає справи за зверненням суб'єкта владних повноважень.

Адміністративний суд розглядає справу, як правило, за місцезнаходженням відповідача, тобто якщо офіційна адреса відповідача зареєстрована на території юрисдикції цього суду.

Керівництво

 Голова суду — Лозовський Олександр Анатолійович
 Заступник голови суду — Мачульський Віктор Володимирович
 Керівник апарату суду — Тарасюк Ольга Олександрівна

Структура
Суд очолює його голова, який має заступника. Правосуддя здійснюють 15 суддів.

Організаційне забезпечення діяльності суду здійснює апарат, очолюваний керівником апарату, який має двох заступників.

До патронатної служби входять помічники суддів. Секретарі судового засідання безпосередньо підпорядковані керівнику апарату та судді, з яким працюють відповідно до внутрішнього розподілу обов'язків.

Апарат суду має 3 відділи.

Відділи

 Управління персоналом
 Організаційного та документального забезпечення діяльності суду, аналітично-статистичної роботи
 Бухгалтерського обліку, управління фінансовими та матеріально-технічними ресурсами

Примітки

Засновані в Україні 2008
Суди Волинської області
Суди першої інстанції
2008 у Волинській області

redpajama_set_name: RedPajamaWikipedia


text:
Q: Close WPF dialog without user interaction I would like to show dialog during some IO operations and after it, close the dialog with code.
Something like:
DialogWindow dialog = new DialogWindow();
dialog.ShowDialog();
.... do some file operation....
dialog.Close();

but dialog won't close. I have to close it by myself. Is here way how to close it with code?

A: DialogWindow dialog = new DialogWindow();
Task.Run(()=>
{
    .... do some file operation....
    Dispatcher.BeginInvoke((Action)dialog.Close);
});
dialog.ShowDialog();

A: Try to use .Show(), not ShowDialog(). If you use ShowDialog, the new window is modal, so you must close manually the window, before you can reach the line of codice dialog.Close().

A: You can not use ShowDialog() because it blocks the current thread.
You could disable the owner window while the process is running and then enable it afterwards like this ('this' is the owner window) (you'll have to be carefull with errors and exceptions, of course):
    async private void Button_Click(object sender, RoutedEventArgs e)
{
  Window1 win = new Window1();
  this.IsEnabled = false;
  win.Show();
  await Task.Run(() =>
    {
      Thread.Sleep(2000);
    });

  win.Close();
  this.IsEnabled = true;
}

An alternative is to run the file operation in the opened dialog (opened with ShowDialog() then) instead of in the main window.

A: If using ShowDialog() then can use below code to close dialog.
(this.Parent as Window).Close();

redpajama_set_name: RedPajamaStackExchange


CRANAplus Bush Support Service Psychologists are visiting as many areas as possible to promote best-practices in managing stress and having difficult conversations with distressed patients and clients in May. This service is to assist communities in coping with the social and emotional impact of the severe drought in NSW.
This entry was posted in Events, News, Uncategorized and tagged drought, mental health, psycology, support on April 17, 2019 by RDA Central West.
It's not just farmers feeling the effects of this on-going drought, but also farming contractors and local rural businesses. Currently, Vinnies, the Salvos, the CWA and Lions are distributing $3,000 cash injection grants to many in need.
Please note you do not have to be a primary producer to apply for these grants. Ag-related businesses (for example, farm machinery mechanics and fencers) are also eligible.
This grant will remain open until funds are exhausted. The closure date will be announced on the CWA website.
This entry was posted in Grants, News and tagged assistance, businesses, drought, farmers on February 5, 2019 by RDA Central West.
The University of Canberra is looking for participants in its Regional Wellbeing Survey.
The survey's mission is to support research that improves the wellbeing and quality of life of people living in rural and regional Australia.
This entry was posted in Events, Uncategorized and tagged drought, University of Canberra, wellbeing survey on November 19, 2018 by RDA Central West.
The NSW Government has established a central location for all information, grants, subsidies and assistance for farmers and small businesses suffering from the drought.
This entry was posted in Grants, News, Uncategorized and tagged drought, farmers, NSW, subsidies, support on August 29, 2018 by RDA Central West.
The Western NSW, NSW Chamber of Business is asking for drought affected businesses in NSW to fill out this short survey so it can formally advocate on behalf of those who are struggling with the dry.
This entry was posted in Grants, News and tagged drought, NSW Business Chamber, survey on August 6, 2018 by RDA Central West.
The CWA is offering financial assistance to help drought affected families meet household expenses. Up to a maximum of $3,000 per family/household, is available to eligible applicants. Expenses can include grocery bills, vehicle maintenance, school, electricity, rates, telephone, dental and medical (gap payments or cost of travel to access treatment) etc. For expenses that are current and/or outstanding.
This entry was posted in Grants and tagged CWA, drought, grants on July 17, 2018 by RDA Central West.
Applications are open for organisations wishing to apply to the Federal Government's Financial Crisis and Material Aid, Emergency Relief and Food Relief funds.
Under the Government's Families and Communities program, grants are available to organisations wishing to apply through a competitive process.
This entry was posted in Grants and tagged drought, emergency relief, farmers, food relief, poverty on July 17, 2018 by RDA Central West.


Tax Manager or Tax Assistant Manager with leading firm in Peterborough.
Hays are working with a leading accountancy practice to help them source a Full Time Tax Senior/Assistant Manager or Manager to manage a portfolio of personal and business tax clients ensuring compliance with HMRC and in-house deadlines.
The successful candidate should ideally have experience of preparing tax computations and returns for Manager/Partner review, reviewing tax work prepared by non-tax specialists and the provision of advice on tax liabilities, working within tax law and HMRC provisions.
We would be keen to hear from individuals with a reasonable level of tax experience. The role will also be supporting the Tax Partner to assist in the provision of tax advisory services across the Peterborough region.
There is scope for upward progression and involvement in business development activities to help raise the profile of the firm. This position would also suit a newly qualified accountant or tax specialist looking to specialise in Taxation. Training will be provided to the suitable candidate, we are therefore looking for a motivated individual who is keen to learn.
Please apply on line or contact Cara Whyte at Hays for more details.