Abstract
BackgroundLimited intrinsic healing potential of the meniscus and a strong correlation between meniscal injury and osteoarthritis have prompted investigation of surgical repair options, including the implantation of functional bioengineered constructs. Cell-based constructs appear promising, however the generation of meniscal constructs is complicated by the presence of diverse cell populations within this heterogeneous tissue and gaps in the information concerning their response to manipulation of oxygen tension during cell culture.MethodsFour human lateral menisci were harvested from patients undergoing total knee replacement. Inner and outer meniscal fibrochondrocytes (MFCs) were expanded to passage 3 in growth medium supplemented with basic fibroblast growth factor (FGF-2), then embedded in porous collagen type I scaffolds and chondrogenically stimulated with transforming growth factor β3 (TGF-β3) under 21% (normal or normoxic) or 3% (hypoxic) oxygen tension for 21 days. Following scaffold culture, constructs were analyzed biochemically for glycosaminoglycan production, histologically for deposition of extracellular matrix (ECM), as well as at the molecular level for expression of characteristic mRNA transcripts.ResultsConstructs cultured under normal oxygen tension expressed higher levels of collagen type II (p = 0.05), aggrecan (p < 0.05) and cartilage oligomeric matrix protein, (COMP) (p < 0.05) compared to hypoxic expanded and cultured constructs. Accumulation of ECM rich in collagen type II and sulfated proteoglycan was evident in normoxic cultured scaffolds compared to those under low oxygen tension. There was no significant difference in expression of these genes between scaffolds seeded with MFCs isolated from inner or outer regions of the tissue following 21 days chondrogenic stimulation (p > 0.05).ConclusionsCells isolated from inner and outer regions of the human meniscus demonstrated equivalent differentiation potential toward chondrogenic phenotype and ECM production. Oxygen tension played a key role in modulating the redifferentiation of meniscal fibrochondrocytes on a 3D collagen scaffold in vitro.
Highlights
Limited intrinsic healing potential of the meniscus and a strong correlation between meniscal injury and osteoarthritis have prompted investigation of surgical repair options, including the implantation of functional bioengineered constructs
When cells reached sub-confluence, first passage (P1) cells were detached with trypsin-EDTA (0.05% w/v) and split 1:2 in order to produce passage 2 cells (P2) that were expanded for another doubling to produce a final monolayer culture of passage 3 (P3) cells
Collagen type II expression, a hallmark of chondrocytic lineage, is significantly depressed following monolayer expansion under hypoxic conditions, and approaches significance in the group of inner meniscal fibrochondrocytes (MFCs) cultured under normal oxygen tension (p = 0.06)
Summary
Limited intrinsic healing potential of the meniscus and a strong correlation between meniscal injury and osteoarthritis have prompted investigation of surgical repair options, including the implantation of functional bioengineered constructs. Avascularity of the inner 2/3rd of the meniscus is associated with limited healing potential of this zone [8,9], and presents a significant clinical problem considering the high incidence of meniscal injury [10,11] Both traumatic injury and surgical resection of total or partial meniscal tissues correlate strongly with the progression of symptomatic osteoarthritis [12,13,14,15]. Recent understanding of this association promotes the sparing of meniscal tissue through arthroscopic partial meniscectomy alone, or in combination with treatment options including allograft transplantation [16], trephination [17], synovial debridement [18,19], and replacement of excised tissues with engineered constructs [20,21]. Knowledge gaps concerning the cell physiology of MFCs and their response to culture microenvironments applied in tissue engineering remain to be investigated
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