Abstract
BackgroundHere, we describe the design and characterization of a novel, cryopreserved, viable osteochondral allograft (CVOCA), along with evidence that the CVOCA can improve outcomes of marrow stimulation for articular cartilage repair.MethodsHistological staining was performed to evaluate the CVOCA tissue architecture. CVOCAs were tested for the presence of extracellular matrix (ECM) proteins and chondrogenic growth factors using ELISA. Cell viability and composition were examined via live/dead staining, fluorescence-activated cell sorting (FACS) analysis, and immunofluorescence staining. FACS analysis and a TNF-α secretion bioassay were used to confirm the lack of immunogenic cells. Effects of the CVOCA on mesenchymal stem cells (MSCs) were tested using in vitro migration and chondrogenesis assays. The ability of the CVOCA to augment marrow stimulation in vivo was evaluated in a goat model.ResultsA method of tissue processing and preservation was developed resulting in a CVOCA with pores and minimal bone. The pores were found to increase the flexibility of the CVOCA and enhance growth factor release. Histological staining revealed that all three zones of hyaline cartilage were preserved within the CVOCA. Chondrogenic growth factors (TGF-β1, TGF-β3, BMP-2, BMP-4, BMP-7, bFGF, IGF-1) and ECM proteins (type II collagen, hyaluronan) were retained within the CVOCA, and their sustained release in culture was observed (TGF β1, TGF-β2, aggrecan). The cells within the CVOCA were confirmed to be chondrocytes and remained viable and functional post-thaw. Immunogenicity testing confirmed the absence of immunogenic cells. The CVOCA induced MSC migration and chondrogenesis in vitro. Experimental results using devitalized flash frozen osteochondral allografts revealed the importance of preserving all components of articular cartilage in the CVOCA. Goats treated with the CVOCA and marrow stimulation exhibited better repair compared to goats treated with marrow stimulation alone.ConclusionsThe CVOCA retains viable chondrocytes, chondrogenic growth factors, and ECM proteins within the intact architecture of native hyaline cartilage. The CVOCA promotes MSC migration and chondrogenesis following marrow stimulation, improving articular cartilage repair.
Highlights
We describe the design and characterization of a novel, cryopreserved, viable osteochondral allograft (CVOCA), along with evidence that the CVOCA can improve outcomes of marrow stimulation for articular cartilage repair
The subchondral bone is penetrated in order to introduce mesenchymal stem cells (MSCs) from the bone marrow into the lesion to generate repair tissue
The pores provide a favorable chondrogenic microenvironment for autologous MSCs, which are released from bone marrow into the cartilage defect following a marrow stimulation procedure and will reside within the CVOCA pores
Summary
We describe the design and characterization of a novel, cryopreserved, viable osteochondral allograft (CVOCA), along with evidence that the CVOCA can improve outcomes of marrow stimulation for articular cartilage repair. A reparative approach, and osteochondral allografts, a restorative approach, are commonly employed techniques. Marrow stimulation is often employed following debridement to fill a lesion with repair tissue [5, 7]. The subchondral bone is penetrated in order to introduce MSCs from the bone marrow into the lesion to generate repair tissue. The initial blood clot filling the lesion typically becomes fibrocartilage repair tissue, with less type II collagen and reduced mechanical integrity compared to normal, hyaline cartilage [8,9,10]. Cells, and growth factors have been studied for this purpose, but their clinical benefits are not yet clearly defined
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