Three-dimensional growth of fibroblasts on carbon fibre mesh and assessment of biocompatibilty by in vitro and in vivo examination was done. Suitable size carbon fiber mesh after sterilization, placed in six well cell culture plate. The mesh was co-cultured with p-MEF cells. At different time intervals the viability and proliferation of the p-MEF cells was evaluated. The primary objective of this study was biological evaluation of carbon fibre mesh which can be used for creation of three-dimensional scaffolds for tissue engineering. Among the possible forms of implants, fibrous matrices are highly promising for the tissue regeneration by acting as a cell-supporting scaffold. Results of in vitro observations of the morphology p-MEF cells seeded on the surface of carbon fibre mesh shows adhesions and attachment of fibroblasts cells to carbon fibres on day 3 post seeding. They attached firmly and were uniformly spread along the fibres on day 5 postseeding and mostly spindle-shaped and cover almost all their surface on day 7 postseeding and such a spreading of cells indicates good adhesions and biocompatibility of carbon fibres. In vivo examination of retrieved sample on day 30 post implantation shows that carbon fibre mesh was covered by dense thick fibrous connective tissue.
Carbon fiber (CF) consists of a multitude of unique physical, chemical and biological characteristics that can be utilized and exploited for a number of diverse applications. Being light weight, high strength, and chemically stable, so they are applied in various fields including aeronautical science and space science. Investigation of applications of carbon fibers to biomaterials was started 30 or more years ago, and various products have been developed. It is used widely in imaging equipment structures to support limbs being X-rayed or treated with radiation.
Carbon fiber is frequently supplied in the form of a continuous tow wound onto a reel. The tow is a bundle of thousands of continuous individual carbon filaments held together and protected by an organic coating, or size, such as polyethylene oxide (PEO) or polyvinyl alcohol (PVA). Each carbon filament in the tow is a continuous cylinder with a diameter of 5–10 micrometers and consists almost exclusively of carbon. The earliest generation had diameters of 16–22 micrometers and later fibers have diameters that are approximately 5 micrometers. Precursors for carbon fibers are polyacrylonitrile (PAN), rayon and pitch.
The latest technological progress has realized nanolevel control of carbon fibers, applications to biomaterials have also progressed to the age of nanosize. Carbon fibers with diameters in the nanoscale (carbon nanofibers) dramatically improve the functions of conventional biomaterials and make the development of new composite materials possible. Carbon nanofibers also open possibilities for new applications in regenerative medicine and cancer treatment. The first three-dimensional constructions with carbon nanofibers have been realized, and it has been found that the materials could be used as excellent scaffolding for bone tissue regeneration. We have developed an innovative approach for the use of CF as a scaffold in the repair of tendon and ligaments and as a suture material for repair of hernial ring.
Carbon as an inert element has advantages over other materials because it is a basic constituent of tissues. The high proportion of tissues of living organisms is composed of carbon compounds so it should be tolerated by the tissues. Over the past 25 years various carbon materials have been investigated in many areas of medicine [1, 2].
The physical and chemical properties of CFs are also determined by their microstructure. This is particularly important in the case of CFs used in medicine. One of the earliest medical uses of CFs was replacement or repair of ligaments and tendons [3, 4, 5]. Most of the studies on carbon fibrous implants confirm that CFs do not inhibit tissue growth, and thus can act as a scaffold for tissue proliferation [3, 6]. Controversy surrounds the mechanism of the disintegration and removal of the implanted CFs. A histological study showed that the CFs gradually broke down and migrated into the nearest lymph nodes with no apparent detrimental effect . In all instances the CFs acted as a scaffold that allowed the regeneration of tendon and ligament. In contrast to these investigations, Morris et al.  showed that fragmentation of fibers did not occur and implant debris was not found in the regional nodes. Moreover, CFs induced significantly more tissue ingrowth than polypropylene mesh at 6–12 months postoperatively. A probable elimination mechanism by erosion of carbon particles and their retention in the fibrous capsule surrounding the implant . Although the results of most investigations of CFs in vivo were evaluated as good, several authors were skeptical as to the superiority of carbon fibrous implants over other prosthetic materials [9, 10]. Carbon fibers were considered to be a good candidate biomaterial for total hip replacement and internal fixation in the form of composites . In spite of controversial results with CFs used for replacement and reconstruction of ligaments and tendons, several other clinical studies were undertaken. The different results obtained in evaluating CFs as a biomaterial can probably be explained by the use of different types of carbon fibers of different physical, structural and chemical properties, resulting from many technological parameters. Most of the papers concerning examinations of CFs for medical purposes describe neither the type of carbon fibers used nor their fundamental properties determining their behavior in a biological environment. We will discuss the use of carbon fibers in biomedical applications under different headings:
- In vitro biocompatibility evaluation
- In vivo biocompatibility evaluation
- Subcutaneous implantation of carbon fibers
- Carbon fibers in repair of abdominal wall defects
- Carbon fiber mesh in reconstruction of abdominal wall defects
- Carbon fibers for gap repair of flexor tendons
- Clinical applications
2. In vitro biocompatibility evaluation
Tissue engineering is the process of creating functional three dimensional (3-D) tissue by combining cells with scaffolds to facilitate cell growth, organization and differentiation. The most important aspect of tissue engineering is the adhesion and proliferation of cells on scaffold material. Biocompatibility of CFs has been the subject of numerous researches. Some of investigators concluded that CFs induces the growth of new tissue . However, there were also announcements questioning biocompatibility of CFs [13, 14, 15]. The different opinions regarding biocompatibility of CFs may be explained by the use of different types of CFs of different physical, structural and chemical properties, resulting from many technological parameters [15, 16]. It has been demonstrated that the cellular response to the fibrous carbon material depends on the degree of crystallinity of the material; therefore only selected types of CFs are suitable for tissue treatment purposes [16, 17, 18].
The fibroblasts are common cells present in the connective tissue that synthesizes and continuously secretes precursors of extra cellular matrix. Fibroblasts play an important role in regeneration of new tissue due to their growth accelerating property of tissue cells by secreting several growth factors and extra cellular matrix (ECM). Primary mouse embryonic fibroblasts (p-MEFs) have a number of properties making them an attractive cell culture model. Compared to other primary explant cultures they are easy to establish and maintain, proliferate rapidly resulting in large numbers of cells produced from a single embryo within several days. Major histocompatibility complex (MHC) Class II antigens are present on the transplanting cells which is responsible for graft rejection. Fibroblasts lack these surface molecules and this makes them relatively immunologically inert.
In the present study, carbon fiber mesh was cut in desired size and after sterilization, placed in six well cell culture plates. The mesh was co-cultured with p-MEF cells. At different time intervals the viability and proliferation of the MEF cells was evaluated. The primary objective of this study was biological evaluation of carbon fiber mesh which can be used for creation of three-dimensional scaffolds for tissue engineering. Carbon fiber used as scaffold for tissue regeneration could simultaneously serve as a support for drug delivery or biologically active agents which would stimulate the tissue growth. Therefore, in this study, we investigated the behavior of carbon fiber mesh in biological environments and their interaction with cells and tissues under in vitro and in vivo conditions.
2.2. Materials and methods
In vitro tests in cell cultures were performed in Biomaterials and Bioengineering Laboratory, Division of Surgery, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India. Prior to cell culture, the carbon fiber mesh was autoclaved at the temperature of 120°C for 30 min. The primary mouse embryo fibroblasts culture (p-MEF) was done as per standard protocol. The cells obtained were washed twice with DMEM containing antibiotics and were centrifuged at 2500 rpm for 8–10 min. The cells were resuspended in cell growth media (DMEM-Low glucose) containing 10% FBS and antibiotics (Mixture of 100 units/ml penicillin and 100 μg/ml streptomycin). The cells were counted countess cell counting kit (Invitrogen) and plated at an average of 2.2 × 105 cells/cm2 in T-25 flasks. They were maintained at 37°C in a humidified atmosphere of 5% CO2 in CO2 incubator. Day after the primary culture, the spindle shaped fibroblast cells were observed and the non-adherent/dead cells were removed by changing the medium. When the cells attained 80–90% confluency (as assessed by observing under inverted microscope, the cells were passaged into new culture flask. Culture medium was removed and cells were washed twice with HBSS (with antibiotic). The cells were detached from the culture flask by using 2 ml of 0.5% of trypsin. The trypsin activity was stopped by adding equal volume of culture medium containing FBS and flushed properly to get the attached cells in the suspension.
After the primary culture, when adherent cells reached to 90% confluency, they were detached with 0.25% trypsin-EDTA solution. Growth medium i.e. DMEM containing 10% fetal bovine serum was added and mixed properly to get single cell suspension. The carbon fiber mesh was cut into small pieces and washed 4–5 times with antibiotics containing Dulbecco’s Modified Eagle Medium (DMEM) and was placed in wells of the culture plate. The cells were seeded at the rate of 2 × 104 cells/cm.2 It was maintained at 37°C in a humidified atmosphere of 5% CO2 in a CO2 incubator. The growth media was changed after 48 h. The seeded mesh was observed and processed for morphological assessment on day 3, 5 and 7 postseeding. Morphological examination was performed using Scanning Electron Microscopic Examination. The seeded mesh was fixed in 2% glutaraldehyde for SEM examination.
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