Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold. Chondrocytes were isolated from Outerbridge grade 0 and 1 AC from four patients undergoing total knee arthroplasty and embedded within 216 bovine collagen I scaffolds. Scaffolds were incubated in hypoxic (3% O2) or normoxic (21% O2) conditions for 1hr, 21hr and 14 days. Gene expression was determined using Q-rt-PCR for col I/II/X, COMP, SOX9, aggrecan and B actin. Matrix production was determined using glycosaminoglycan (GAG) content relative to cell count determined by DNA quantification. Cell viability and location within the matrix was determined by Live/Dead assay and confocal microscopy. Statistical analysis was performed using a two-tailed T-test.Purpose
Method
The biomechanical role of the meniscus in the knee joint is a function of its extracellular matrix which consists of type I collagen throughout, type II collagen in the inner meniscus region and glycosaminoglynated (GAG) proteins of which aggrecan is the most prevaleet. Meniscus reparative capacity is limited, particularly when a defect is located in the inner avascular portion, and menisectomy predisposes the joint to osteoarthritis. Using meniscus cells in tissue engineering strategies has been advocated to generate functional meniscus substitutes. However, meniscus cells, like chondrocytes of cartilage, lose their matrix-forming phenotype during culture expansion. Co-culture of chondrocytes with stem cells has been shown to result in enhanced matrix formation. We hypothesized that meniscus cells in co-culture with stem cells will result in increased matrix formation. Tissue specimens were obtained after approval of the local ethical committee and informed consent. Menisci were obtained from 3 patients undergoing total knee arthroplasty; (53–84; mean age 66.6). Meniscus cells were isolated after digestion of menisci with collagenase II. Isolated meniscus cells were plated for 24–48 hr before use. Bone marrow aspirates were obtained from the iliac crest of 3 donors: 1 female (46) and 2 males (15 and 21) undergoing routine orthopaedic procedures. Plastic adherent bone marrow stromal cell populations were isolated and expanded under normal oxygen tension of 21%O2 in a-MEM growth media plus FGF-2 until passage 2. Cells were mixed at a variety of meniscus cells (Men): BMSC ratio including 5/95, 10/90 and 25/75, respectively. Mixed cells were centrifuged to form spherical pellets followed by culture in a defined serum free chondrogenic differentiation medium. Control groups were pure Men and pure BMSCs. Total cell number per pellet was 25×104. Pellets were cultured for 3 weeks under normal oxygen tension. Thereafter, pellets were processed: biochemically for GAG and DNA content, and histologically for Safranin-O staining of sulphated GAG and immunohistochemical analyses for collagen types I and II. Analysis was performed on a minimum of 2 independent pellets.Purpose
Method
Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2. Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses.Purpose
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