Validation of Laboratory Test for Predicting Bone Tissue Regeneration
Validation of Laboratory Test for Predicting Bone Tissue Regeneration
The aim of the study is to determine whether the evaluation of pro-osteogenic activity of
 autologous serum may predict the effectiveness of platelet gel in regenerating bone tissue in
 patients with nonunions of long bones.

 Serum samples will be collected before the surgery, and their pro-osteogenic activity will be
 evaluated by using a bioactivity test (mineralization assay).

 In order to determine whether the pro-osteogenic activity of the serum is able to
 discriminate between individuals who will achieve or will not achieve bone consolidation, the
 laboratory results will be correlated with clinical and radiographic results at 12 months,
 when patients will be considered as healed or not healed.
Pseudarthrosis (or 'nonunions') is defined as a bone movement which occurs after about 6
 months from the traumatic event as consequence of the inadequate healing of a fracture. The
 use of regenerative medicine for treating recalcitrant pseudarthrosis is considered a
 promising strategy to promote bone consolidation. Among the various approaches used, the
 local cell therapy based on autologous mesenchymal stromal cells (MSC), combined with or
 without growth factors, has been extensively used. MSC reside within the stromal compartment
 of bone marrow which may be obtained from the iliac crest. Growth factors may be discharged
 from platelet gel (PG) which is obtained after activation of autologous platelet
 concentrates. Nevertheless, the results of recent clinical studies show that the above
 mentioned regenerative approach shows a failure rate of approximately 30% (Calori et al,
 2008). The availability of a laboratory test which may predict the regenerative capacity of
 the PG may help the orthopaedic surgeon in addressing the decisions regarding the
 regenerative approach, e.g. to use or non use autologous platelet concentrates or to enhance
 the use of autologous derivatives with recombinant factors.The rationale for the use of
 platelet concentrates in pseudarthrosis is the significant reduction of osteoinductive growth
 factors observed at the lesion site (Gandhi et al., 2005). Platelet gel mimics what happens
 physiologically after any bone injury, when platelets are entrapped in a clot within a fibrin
 matrix. Platelet activation determines the release of various growth factors promoting bone
 healing. The optimal level and ratio of growth factors and their synergistic effects should
 be more efficient than single recombinant molecules, even though no data are available on the
 most relevant molecules and on their optimal amount. In this context, the in vitro
 mineralization assay could be a valid tool to assess the pro-osteogenic activity of platelet
 gel, thus obviating the measurement of the myriad of platelet-derived substances.

 The aim of the study is to determine whether the evaluation of pro-osteogenic activity of
 autologous serum may predict the effectiveness of PG in the regeneration of bone tissue in
 patients with nonunions of long bones. The rationale is based on the following issues:

 -  in 30% of long bone nonunions treated with PG, bone consolidation is not obtained
 (Calori et al, 2008);

 -  the proportion of growth factors in serum is comparable to that observed in platelet gel
 after activation, even if the concentration is lower;

 -  the autologous serum is able to support the 'in vitro' osteogenic differentiation of
 MSCs and the formation of mineral nodules (mineralization assay) (Granchi et al, 2010);

 -  the correlation between 'in vitro' mineralization and regenerative capacity has been
 demonstrated in various experimental models (De Bari et al, 2008);

 -  preliminary data obtained in our lab showed that in patients affected by congenital
 pseudarthrosis of the tibia the presence of osteoinductive growth factors in serum is an
 essential requirement to warrant the effectiveness of the local cell therapy with MSC
 and PG (submitted paper).

 In order to evaluate the ability of the autologous serum in inducing the mineralization in
 vitro the investigators will use a bioactivity test (mineralization assay). For this purpose
 serum samples of patients candidates to the treatment with MSC and PG will be collected the
 day before the surgery and used for cultivating normal MSC, whose ability to mineralize in
 vitro will be previously verified in standard culture conditions.

 Further analysis will be conducted in order to determine whether the pro-osteogenic
 properties are maintained over time, and serum samples will be collected the 1 month before
 the surgery.

 In order to determine whether the pro-osteogenic activity of the serum is able to predict and
 discriminate between individuals who achieve or not achieve bone consolidation, the
 laboratory results will be correlated with clinical and radiographic results at 12 months,
 when patients will be considered as healed or not healed.
Eligible patients will be identified according to the following criteria.

 Inclusion criteria:

 -  patients with aseptic nonunion of long bones and candidates for treatment with
 platelet gel and MSC in addition to fixation devices and bone grafting;

 -  patients who have given their written consent to participate in the study.

 Exclusion criteria:

 -  patients with bone infections;

 -  patients with congenital disorders involving the skeletal development;

 -  patients treated with corticosteroids or other immunosuppressive agents;

 -  patients suffering from mental or psychiatric disorders that preclude the possibility
 of correctly adhering to the protocol.