May S. Jacobson

Activation of Platelet-Rich Plasma Using Soluble Type I Collagen

PURPOSE Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important to oral tissue healing. But application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation through the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this work, our hypothesis was that soluble type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and stimulating growth factor release from the platelets and granulocytes. MATERIALS AND METHODS PRP from human donors was clotted using type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of platelet-derived growth factor (PDGF)-AB, transforming growth factor (TGF)-beta1, and vascular endothelial growth factor (VEGF) from both types of clots was measured over 10 days using enzyme-linked immunosorbent assasy. RESULTS Clots formed using type I collagen exhibited far less retraction than those formed with bovine thrombin. Bovine thrombin and type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-beta1 during the first 5 days after activation. CONCLUSIONS The use of type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF compared with currently available methods of clot activation.

Platelet Activation by Collagen Provides Sustained Release of Anabolic Cytokines

Background: Platelet-rich plasma (PRP) has been increasingly used in sports medicine applications. Platelets are thought to release growth factors important in wound healing, including transforming growth factor (TGF-β1), platelet-derived growth factor (PDGF-AB), and vascular endothelial growth factor (VEGF). However, little is known about the effect of platelet activator choice on growth factor release kinetics. Hypothesis: The choice of platelet activator would affect the timing and level of growth factor release from PRP. Study Design: Controlled laboratory study. Methods: Platelet-rich plasma aliquots were activated with either thrombin or collagen. A control group of whole blood aliquots was clotted with thrombin. Supernatant containing the released growth factors was collected daily for 1 week. Levels of TGF-β1, PDGF-AB, and VEGF were measured using enzyme-linked immunosorbent assay (ELISA). Results: The use of thrombin as an activator resulted in immediate release of TGF-β1 and PDGF-AB, while the collagen-activated PRP clots released similar amounts each day for 5 days. The use of collagen as an activator resulted in an 80% greater cumulative release of TGF-β1 from the PRP aliquots over 7 days (P < .001). Concentrating platelets to 3 times the systemic blood level resulted in a 3-fold higher release of TGF-β1, 2.5-fold greater release of PDGF, and 5-fold greater release of VEGF (all P < .0001) when compared with whole blood control clots, but no significant differences in the timing of release were noted. Conclusion: These experiments demonstrated that the choice of platelet activator can significantly influence the release kinetics of cytokines from PRP, with thrombin resulting in an immediate release and collagen having a more sustained release pattern. Clinical Relevance: The level and rate of growth factor release depends on the selected platelet activator, a factor that should be considered when selecting a PRP system for a given application.

Bone Marrow Concentrate: A Novel Strategy for Bone Defect Treatment

BACKGROUND Although strong efforts have been made over the last decade to introduce stem cell and tissue engineering treatment strategies to the field of orthopaedics, only few clinical applications are currently available. MATERIALS AND METHODS The clinical outcomes of ten patients with volumetric bone deficiencies treated with mesenchymal stem cells and bone marrow aspirate are presented in this case series. Results were evaluated with radiographs. In addition to the in vivo data, we also presented in vitro data of BMC cultivated onto a porous collagen I scaffold and the technique of bone marrow aspiration via a commercially available system. RESULTS Our results demonstrated that there is a rationale for a clinical application of BMC / bone aspirate in the treatment of osseous defects. The intraoperative harvest procedure is a safe method and does not significantly prolong the time of surgery. In addition, MSC isolated from the aspirate was able to adhere and proliferate onto a collagen scaffold in significant numbers after a 15 min incubation period. These cells were then able to allow osteogenic differentiation in vitro without any osteogenic stimuli. CONCLUSIONS The local application of BMC / bone aspirate in the treatment of bone deficiencies may be a promising alternative to autogenous bone grafting and help reduce donor site morbidity.

Concentration of bone marrow total nucleated cells by a point-of-care device provides a high yield and preserves their functional activity.

Stem and progenitor cell therapy is a novel strategy to enhance cardiovascular regeneration. Cell isolation procedures are crucial for the functional activity of the administered cellular product. Therefore, new isolation techniques have to be evaluated in comparison to the Ficoll isolation procedure as the current gold standard. Here we prospectively evaluated a novel point-of-care device (Harvest BMAC System) for the concentration of bone marrow total nucleated cells (TNC) in comparison to the Ficoll isolation procedure for bone marrow mononucleated cells (MNC). The yield in total numbers of TNC was 2.4-fold higher for Harvest compared to Ficoll. Despite significant differences in their cellular compositions, the colony-forming capacity was similar for both products. Intriguingly, the migratory capacity was significantly higher for the Harvest TNC (164 ± 66%; p = 0.007). In a mouse model of hind limb ischemia, the increase in blood flow recovery was similar between Harvest BM-TNC and Ficoll BM-MNC (0.53 ± 0.20 vs. 0.46 ± 0.15; p = 0.88). However, adjustment of the injected cell number based on the higher yield of Harvest TNC resulted in a significant better recovery (0.64 ± 0.16 vs. 0.46 ± 0.15; p = 0.003). Cells concentrated by the Harvest point-of-care device show similar or greater functional activity compared to Ficoll isolation. However, the greater yield of cells and the wider range of cell types for the Harvest device may translate into an even greater therapeutic effect.

An in vitro and in vivo evaluation of autologous platelet concentrate in oral reconstruction.

A platelet concentrate, when combined with calcified thrombin, produces a platelet gel that has been used to achieve hemostasis and modulate bone growth and wound healing. The recovery of high concentrations of viable platelets and their resulting growth factor levels represents the most important factor in the clinical utility of a platelet concentrate because only functional platelets can release the growth factors that are necessary to induce tissue growth and bone regeneration. The SmartPReP system’s efficiency in recovery of platelets from a sample of whole blood averaged 70.6%, almost twice that of various manual techniques using laboratory centrifuges. Platelet concentrates prepared by the SmartPReP system had a viability equal to platelet concentrates prepared for transfusion as measured by hypotonic stress, platelet aggregation, and p-selectin. A series of clinical case studies demonstrates the use of autologous platelet gel in oral surgery.

Platelets, but not erythrocytes, significantly affect cytokine release and scaffold contraction in a provisional scaffold model

Platelets and erythrocytes are major components of wound provisional scaffolding. In this study, we hypothesized that the concentration of platelets and erythrocytes would significantly affect fibroblast‐mediated contraction of three‐dimensional scaffolds or the release of cytokines from the scaffold. To test this hypothesis, human anterior cruciate ligament fibroblasts were cultured in one of four scaffolds: a collagen matrix, a collagen‐fibrin matrix containing the same concentration of platelets as whole blood, a collagen‐fibrin matrix containing a high platelet concentration, and a collagen–fibrin matrix containing a high platelet concentration and red blood cells. Cytokine release from the four groups of gels and gel contraction were measured over a 10‐day period. The results of these assays supported greater cytokine release, fibroblast proliferation, and gel contraction in scaffolds with higher platelet concentration. In contrast, the addition of erythrocytes did not significantly stimulate or suppress scaffold contraction or growth factor release from the provisional scaffolds. We concluded that while platelet concentration can significantly impact cytokine release and scaffold retraction in a provisional scaffold, the inclusion of erythrocytes does not have a significant effect on these same behaviors. Therefore, while platelets may be an important regulator of repair processes after injury, it is less likely that erythrocytes have a similar function.

Effects of platelet rich plasma and acellular bone marrow on gene expression patterns and DNA content of equine suspensory ligament explant cultures

REASONS FOR PERFORMING STUDY Suspensory ligament (SL) desmitis is a common source of lameness. The results of this study will determine if blood-derived products stimulate SL matrix synthesis and have potential as regenerative therapies for SL desmitis OBJECTIVES To determine if various blood-based biological products including plasma, blood, PRP, platelet poor plasma (PPP) and ABM aspirate stimulates anabolic and/or catabolic pathways in suspensory ligaments (SL). METHODS The body of the SL was harvested from 6 horses and used to establish explant cultures. Explants were cultured in plasma, blood, PRP, PPP or ABM at concentrations of 10, 50 or 100%. Anabolic responses were assessed by use of quantitative PCR for collagens type I and III, cartilage oligomeric matrix protein (COMP) and decorin. Total DNA and collagen protein content were also measured. Catabolic reactions were measured by quantitative PCR for matrix metalloproteinases 3 and 13 (MMP-3, MMP-13). RESULTS Acellular bone marrow aspirate at 100% stimulated decorin and COMP mRNA synthesis more than all other treatments at all concentrations. No treatment at any concentration stimulated the catabolic gene MMP-13; only 50% ABM stimulated MMP-13 mRNA expression. CONCLUSIONS Acellular bone marrow is indicated, and might be preferred to plasma, blood or PPP, as a blood-based biological source for SL tissue regenerative therapy. Long-term, placebo controlled case studies are indicated to determine if ABM aids in recovery from SL desmitis. POTENTIAL RELEVANCE Bone marrow aspirate is an autogenous, readily available biological source for SL regenerative therapy where the aim is to stimulate matrix synthesis.

Evaluation of an automated system for the collection of packed RBCs, platelets, and plasma

BACKGROUND: This study evaluated the quality of WBC‐reduced platelets, RBCs, and plasma collected on a new system (Trima, Gambro BCT) designed to automate the collection of all blood components. The study also evaluated donor safety and suitability of these components for transfusion.

A Prospective Analysis of Cholestasis in Infants Supported with Extracorporeal Membrane Oxygenation

Summary: Cholestasis develops in many infants supported with extracorporeal membrane oxygenation. We prospectively investigated the role of hemolysis and di-(2-ethylhexyl) phthalate exposure in the development of this cholestasis. Both di-(2-ethylhexyl) phthalate levels and hemolysis, as measured by maximum free hemoglobin, were significantly (p < 0.025) associated with the degree of cholestasis. Other clinical and laboratory factors that may contribute to cholestasis were also investigated and not found to be related to the degree of cholestasis. We speculate that hemolysis during extracorporeal membrane oxygenation support produces a large bilirubin load whose excretion is inhibited by mechanisms similar to the inspissated bile syndrome and/or by di-(2-ethylhexyl) phthalate. This would result in a predominantly direct hyperbilirubinemia with little evidence of hepatocellular or canalicular injury.

An In Vitro Evaluation of a New Plasticizer for Polyvinylchloride Medical Devices

The rate of Hatcol‐200 (H‐200) leaching from polyvinylchloride (PVC) by serum is one‐hundredth that of di‐2‐ethylhexyl phthalate (DEHP) as determined by radioassay. There is a proportional decrease in leach‐ability of DEHP but not of H‐200 as the plasma proteins are diluted. Saline extracts of DEHP demonstrated a low but progressive rate of leaching with an accumulation of 11 μg/ml after seven weeks whereas H‐200 showed only a constant residue of 0.35 μ/ml. Normal human serum incubated for three weeks at 37 C with PVC strips plasticized with DEHP produced significant growth‐inhibition of human diploid fibroblasts. Human serum incubated in an identical manner with PVC plasticized with H‐200 for up to four weeks demonstrated no effect on tissue culture. These studies indicate that H‐200 deserves further evaluation as a replacement for DEHP in PVC biomedical devices.