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Platelet Optimization and Characterization
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Collaborating with investigators from The Center for Blood Research, The Sports Medicine Research Lab is researching the effect of various conditions on platelet activation for ACL and meniscal repair.
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The provisional scaffold, which fills in the wounds site in injured tissues found outside the joint (extra-articular tissues), is subject to premature failure for tissues found inside the joint (intra-articular tissues). This is likely a key contributor to the failure of these tissues to heal (non-union) (Fig 1).
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For that reason, we are studying the effects of placing a substitute collagen-platelet scaffold in the wound site of an intra-articular model tissue, the ACL, in an effort to stimulate intra-articular healing of this tissue after complete transection.
Our theory is that cellular and vascular invasion of the wound site is dependent on the platelet concentration in the collagen-platelet scaffold, and that the differences in cell vascular invasion will alter the strength of the healing tissue. Proving this could significantly alter the way these injuries are approached, studied and treated.
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Preliminary work has shown a collagen-platelet scaffold can stimulate histologic healing and return of biomechanical strength in a complete ACL transection model.
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Work in platelet optimization will define the effect of platelet concentration in the wound site on the functional healing of the ACL, and explore two potential mechanisms for platelet effects on wound healing, including the stimulation of cellular and vascular invasion into the wound site.
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| click for larger image |
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The ideal provisional scaffold would:
- provide an environment for development of a viable, cellular tissue which adheres intimately with the surrounding tissue,
- increase in strength with time,
- be capable of repairing damage as it occurs.
To accomplish this the provisional scaffold should initially:
- integrate with the host tissue,
- support the gradual ingrowth of surrounding cells,
- supply nutritional needs via diffusion or vascular invasion,
- facilitate cellular extracellular matrix production and organization to strengthen the repair site.
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The healing process in extra-articular tissues needed to accomplish these goals is complex.
The process starts with primary hemostasis and formation of a platelet plug. The platelets release over twenty known cytokines in a sequential fashion that stimulates the surrounding cells, induces chemotaxis and spurs the release of other growth factors and ECM molecules.
Adding one or two growth factors had only met with limited success in stimulating ligament healing(3, 12, 13), so we elected to use autologous platelets and the myriad of growth factors and proteins they express to stimulate the healing process in the intra-articular defect.
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The role for growth factors released by platelets, such as IGF-1, TGF-b, VEGF, PDGF and FGF-2, in the healing of tendon and ligament is widely recognized (7). ACL cell migration in vitro has been stimulated by TGF-b1, while PDGF-AB and FGF-2 can stimulate ACL cell proliferation in a 3-D collagen scaffold (6). PRP is also reported to stimulate healing of bone in both animal models and human lumbar spinal fusion (5). Fibrin glues and platelet gels have been used to improve the strength of the abdominal wall fascia after hernia repair in rats (14).
The use of PRP was found in increase the scar mass and cellular density of the repair tissue. However, the use of platelets to stimulate tendon and ligament healing directly has been less widely reported(1) and is a relatively new field of inquiry.
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Our initial studies focused on the use of a collagen-GAG sponge to facilitate ACL cell migration(9, 10). However, migration rates using this substrate were slow (9, 10). We attempted to stimulate additional cell migration and collagen production using individual growth factors, including TGF-2, FGF-2, PDGF-AB and EGF, and had modest success, with moderate increases cell proliferation and collagen production rates in the scaffold (6, 8). However, we felt the in-growth was inadequate and that adhesion between the explanted tissue and scaffold was poor. We next elected to study the use of a collagen gel. Adhesion as well as rates of cell migration into the gel were significantly improved with this form of collagen (11) (2).
Left with the problem of which growth factors to add to the provisional scaffold to stimulate healing, we examined the wound healing process in other tissues. It became clear that this is a complex process starting with platelet activation. The platelets then begin to sequentially release a variety of growth factors and proteins, which summon additional repair cells to the wound site (including neutrophils and macrophages).
Rather than trying to design a de novo implant which contained all of the factors required for the healing process at the right concentrations to be released at the correct times, we elected to work towards stabilizing the platelet-fibrin plug for the intra-articular environment and allowing the healing cascade to occur as it does in those tissues that successfully heal.
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We found that mixing the platelets and plasma proteins with an acid-soluble collagen gel resulted in a composite provisional scaffold that was resistant to plasmin degradation in vitro. This is likely due to the fact that collagen requires an MMP cofactor to be degraded and cannot be degraded by plasmin alone when placed into a fibrin matrix (4). An additional benefit of the collagen was that it also activates platelets, triggering the initial phase of the healing cascade. Cell migration assays and cell proliferation assays proved that this combination was fibroinductive for ACL cells, and supported cell proliferation and collagen production(2).
Using these assays, we were able to optimize the percent of PRP that optimally stimulated cell migration, cell proliferation and collagen production, and this collagen-platelet hydrogel is used for our in vivo studies.
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Page references (pdf)
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