s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S153 251 USE OF A SURGICAL HEMOSTAT MODULATES PROPERTIES OF CLOT ASSOCIATED WITH SUBCHONDRAL BONE MICRODRILLING IN AN OVINE MODEL FOR CARTILAGE REPAIR M. Long y, M. Hawkins z. y Stryker Orthobiologics, Mahwah, NJ, USA; z Stryker Joint Preservation, Mahwah, NJ, USA Purpose: Since the properties of the clot formed in microfracture therapy are influential to clinical outcome, we aimed to evaluate whether the use of a surgical hemostat (SH) to control bleeding and augment and stabilize the microfracture clot would improve the clot properties in the cartilage defects. Methods: Twenty sheep were operated and cared for at Thomas D. Morris (Reisterstown, MD) under IACUC approval #12-038 and GLP quality controls. Two full thickness chondral defects were created in the distal part of the femur: one 8mmx10mm in the medial femoral condyle; one 6mmx12mm in the trochlea. Each defect was treated by microfracture/microdrilling (MFX MicroFX OCD, Stryker) and alternatively treated with a surgical hemostat containing autologous plasma, pepsinized microfibrillar collagen and recombinant thrombin (SH VITAGEL RT Surgical Hemostat, Stryker). The hemostat was prepared individually for each treated defect per manufacturer’s instructions for use. SH was applied to the defect once a bleeding bed was achieved at the bottom of the defect in sufficient volume (z0.20.4cc) to fill the entire defect, and was then allowed to congeal. Time to hemostasis when bleeding stopped was measured starting at the time when the hemostat was applied. The joint was closed once bleeding / oozing was completely stopped. The sheep were placed in a Thomas splint for 2 week post-op. Clot/cartilage fill was visually evaluated (noninstrumented). Clot/cartilage stiffness was assessed semi-quantitatively using a flexible #10 scalpel blade probewith a 0-4 scoring relative to the surrounding intact cartilage. Time 0 and 2 weeks clots were carefully harvested from the defects and sliced in three samples, when allowed by the size of the clot samples, for subsequent histology/histochemistry, biochemistry (ELISA for GAG, COL I & II), and DNA/Cellularity (RT-PCR for COL I & II, Aggrecan) evaluations. For clot and cartilage histology/ histochemistry, frozen and decalcified sections, respectively, were processed and slides stained with HE y. y INIBICUniv. Hosp. of A Coru~ na (CHUAC), A Coru~ na, Spain; z INIBIC Dept. of Med., Univ. of A Coru~ na, A Coru~ na, Spain; xDept. of Med. Specialties, Univ. of Alcal a de Henares, Madrid, Spain Purpose: The purpose was to study chondrogenesis of adult human Mesenchymal Stromal Cells, isolated from bone marrow (hBMSCs), when culture on collagen (Col) biomaterials and to evaluate the suitability of the neotissue formed to use in Tissue Engineering. Methods: hBMSCswere culturedduring30days inchondrogenicmedium with Transforming growth factor b-3 (TGFb-3) on the following biomaterials: Col I, Col IþCol II, Col Iþ heparan sulphate (HS), Col IþCol IIþ heparan sulphate (HS), Col IþCol IIþ chondroitin sulphate (CHS) and Col Iþ heparin (OLH3). We have tested growth and cell morphology on the constructs by histochemical and Scanning (SEM) and Transmission (TEM) Electron Microscopy. Chondrogenic differentiationwas evaluated in all the constructs by histochemical and immunohistochemistry analyses and molecular biology.Moreover, Col concentrationwasmeasured in supernatants along the culture. Results: Histology showed that hBMSCs have grown through the scaffolds, being the cell percentage respect to the total area of the scaffold: >75% in Col I, Col IþCol II, Col IþHS, Col IþCol IIþHS, Col IþCol IIþCHS;< 50% in Col IþOLH3. It was observed a big amount of extracellular matrix (ECM), except in Col IþOLH3. ECM from Col IþCol II and Col IþHS showed the higher metachromasia for Safranin O proteoglycan staining, whereas Col I, Col IþCol IIþHS, Col IþCol IIþCHS showed an intermediate metachromasia (Figure). Col II immunostainingwas highly positive in Col I and Col IþHS ECM and positive intracellularly in Col IþCol II, Col IþCol IIþ HS, Col IþCol IIþCHS (Figure). Col IþOLH3 did not show positivity for proteoglycans and Col II. By measurement of relative expression levels (REL), COL II gene expression was seen in cells all over the biomaterials, Col I (0.63 REL), Col IþCol II (1.00 REL), Col IþHS (0.47 REL), Col IþCol IIþHS (0.93 REL), Col IþCol IIþCHS (3.57 REL) and Col IþOLH3 (5.20 REL). AGG gene expressionwas higher in Col IþCol IIþHS (6.47 REL) constructs than in Col I (0.00 REL), Col IþCol II (0.95 REL),Col IþHS (0.17 REL), Col IþCol IIþCHS (0.00 REL) and Col IþOLH3 (0.00 REL). TEM analysis showed a big amount of mitochondria and oval/rounded shape cells (Figure). We could also see oval/rounded cells and ECM by SEM. Col released (measured as mg/total volume) was detected in all the supernatants, finding the highest concentration in Col IþHS biomaterials cultures except at day 21: Col I (137.33), Col IþCol II (0.00), Col IþHS (0.00), Col IþCol IIþHS (3.33), Col IþCol IIþCHS (13.90) and Col IþOLH3 (0.00). Conclusions: Data showed hBMSCs are able to attach and proliferate on all Col biomaterials, except in Col IþOLH3. These cells showed abundant ECM. Analysis showed differentiated cellular phenotype and an ECM similar to cartilage, inbiomaterials composedbyHS. Theneotissue formed in the other biomaterials showed an intermediate phenotype. The neotissue formed in Col and HS biomaterials could be useful for cartilage tissue engineering. Acknowledgements: B. Parma (OPOCRIN S.P.A.); CAM (S2009/MAT-1472); CIBER BBN CB06-01-0040; Red Gallega de Terapia Celular (REDICENT); SAI-UDC; CSR is beneficiary of a fellowship from Diputaci on de A Coru~ na; P. Esbrit (Fundaci on Jim enez Diaz). Figure: Images from the different constructs (columns). Ordered in rows are shown the different analysis: Scanning (SEM) and Transmission (TEM) Electron Microscopy, Safranin O staining (SO) and Collagen type II immunostaining (Col II). 253 MRI MORPHOLOGICAL AND QUANTITATIVE EVALUATION OF KNEE ALLOGRAFT REPAIR AT 3, 6 AND 9 MONTHS POST-OP: EARLY SURVEILLANCE DEMONSTRATES NASCENT PHYSIOLOGICAL INCORPORATION OF ALLOGRAFT MATERIAL IN PAIN FREE PATIENTS J.M. Farber y, B. Holladay z, J. Tamez-Pena x, S. Totterman k, E. Brandser y, K. Baum k. yQmetrics, Cincinnati, OH, USA; zCommonwealth Orthopaedic Ctr.s, Edgewood, KY, USA; x Tec de Monterry, Monteryy, Mexico; kQmetrics,