Hypoxic conditions increase hypoxia-inducible transcription factor 2α and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients

Stem cells derived from the infrapatellar fat pad (IPFP) are a potential source of stem cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we investigated the effects of hypoxia on gene expression changes and chondrogenesis in stem cells from the IPFP removed from elderly patients with osteoarthritis at total knee replacement. Adherent colony-forming cells were isolated and cultured from the IPFP from total knee replacement. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured for 14 days as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. Gene expression analysis, DNA and glycosoaminoglycan assays and immunohistochemical staining were determined to assess chondrogenesis. IPFP-derived adherent colony-forming cells stained strongly for markers of adult mesenchymal stem cells, including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Cell aggregates of IPFP cells showed a chondrogenic response. In hypoxic conditions there was increased matrix accumulation of proteoglycan but less cell proliferation, which resulted in 3.5-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of hypoxia-inducible transcription factor (HIF)2α and not HIF1α, and the expression of key transcription factors SOX5, SOX6 and SOX9, and that of aggrecan, versican and collagens II, IX, X and XI, was also increased. These results show that cells with stem cell characteristics were isolated from the IPFP of elderly patients with osteoarthritis and that their response to chondrogenic culture was enhanced by lowered oxygen tension, which upregulated HIF2α and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of cells derived from the IPFP

The matrix-forming phenotype of cultured human meniscus cells is enhanced after culture with fibroblast growth factor 2 and is further stimulated by hypoxia

Human meniscus cells have a predominantly fibrogenic pattern of gene expression, but like chondrocytes they proliferate in monolayer culture and lose the expression of type II collagen. We have investigated the potential of human meniscus cells, which were expanded with or without fibroblast growth factor 2 (FGF2), to produce matrix in three-dimensional cell aggregate cultures with a chondrogenic medium at low (5%) and normal (20%) oxygen tension. The presence of FGF2 during the expansion of meniscus cells enhanced the re-expression of type II collagen 200-fold in subsequent three-dimensional cell aggregate cultures. This was increased further (400-fold) by culture in 5% oxygen. Cell aggregates of FGF2-expanded meniscus cells accumulated more proteoglycan (total glycosaminoglycan) over 14 days and deposited a collagen II-rich matrix. The gene expression of matrix-associated proteoglycans (biglycan and fibromodulin) was also increased by FGF2 and hypoxia. Meniscus cells after expansion in monolayer can therefore respond to chondrogenic signals, and this is enhanced by FGF2 during expansion and low oxygen tension during aggregate cultures

Human meniscus cells express hypoxia inducible factor-1α and increased SOX9 in response to low oxygen tension in cell aggregate culture

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)α(I) in response to 5% oxygen, and this hypoxia-induced expression of P4Hα(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1α inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1α gene and downstream target genes (namely, those encoding P4Hα(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation

Calcaneal tendon plasticity following gastrocnemius muscle injury in rat

Cross-talk between skeletal muscle and tendon is important for tissue homeostasis. Whereas the skeletal muscle response to tendon injury has been well-studied, to the best of our knowledge the tendon response to skeletal muscle injury has been neglected. Thus, we investigated calcaneal tendon extracellular matrix (ECM) remodeling after gastrocnemius muscle injury using a rat model. Wistar rats were randomly divided into four groups: control group (C; animals that were not exposed to muscle injury) and harvested at different time points post gastrocnemius muscle injury (3, 14, and 28 days) for gene expression, morphological, and biomechanical analyses. At 3 days post injury, we observed mRNA-level dysregulation of signaling pathways associated with collagen I accompanied with disrupted biomechanical properties. At 14 days post injury, we found reduced collagen content histologically accompanied by invasion of blood vessels into the tendon proper and an abundance of peritendinous sheath cells. Finally, at 28 days post injury, there were signs of recovery at the gene expression level including upregulation of transcription factors related to ECM synthesis, remodeling, and repair. At this time point, tendons also presented with increased peritendinous sheath cells, decreased adipose cells, higher Young’s modulus, and lower strain to failure compared to the uninjured controls and all post injury time points. In summary, we demonstrate that the calcaneal tendon undergoes extensive ECM remodeling in response to gastrocnemius muscle injury leading to altered functional properties in a rat model. Tendon plasticity in response to skeletal muscle injury merits further investigation to understand its physiological relevance and potential clinical implications

Oxygen Tension Is a Determinant of the Matrix-Forming Phenotype of Cultured Human Meniscal Fibrochondrocytes

BACKGROUND: Meniscal cartilage displays a poor repair capacity, especially when injury is located in the avascular region of the tissue. Cell-based tissue engineering strategies to generate functional meniscus substitutes is a promising approach to treat meniscus injuries. Meniscus fibrochondrocytes (MFC) can be used in this approach. However, MFC are unable to retain their phenotype when expanded in culture. In this study, we explored the effect of oxygen tension on MFC expansion and on their matrix-forming phenotype. METHODOLOGY/PRINCIPAL FINDINGS: MFC were isolated from human menisci followed by basic fibroblast growth factor (FGF-2) mediated cell expansion in monolayer culture under normoxia (21%O(2)) or hypoxia (3%O(2)). Normoxia and hypoxia expanded MFC were seeded on to a collagen scaffold. The MFC seeded scaffolds (constructs) were cultured in a serum free chondrogenic medium for 3 weeks under normoxia and hypoxia. Constructs containing normoxia-expanded MFC were subsequently cultured under normoxia while those formed from hypoxia-expanded MFC were subsequently cultured under hypoxia. After 3 weeks of in vitro culture, the constructs were assessed biochemically, histologically and for gene expression via real-time reverse transcription-PCR assays. The results showed that constructs under normoxia produced a matrix with enhanced mRNA ratio (3.5-fold higher; p<0.001) of collagen type II to I. This was confirmed by enhanced deposition of collagen II using immuno-histochemistry. Furthermore, the constructs under hypoxia produced a matrix with higher mRNA ratio of aggrecan to versican (3.5-fold, p<0.05). However, both constructs had the same capacity to produce a glycosaminoglycan (GAG) -specific extracellular matrix. CONCLUSIONS: Our data provide evidence that oxygen tension is a key player in determining the matrix phenotype of cultured MFC. These findings suggest that the use of normal and low oxygen tension during MFC expansion and subsequent neo-tissue formation cultures may be important in engineering different regions of the meniscus

Paternal resistance training modulates calcaneal tendon proteome in the offspring exposed to high-fat diet

The increase in high-energy dietary intakes is a well-known risk factor for many diseases, and can also negatively impact the tendon. Ancestral lifestyle can mitigate the metabolic harmful effects of offspring exposed to high-fat diet (HF). However, the influence of paternal exercise on molecular pathways associated to offspring tendon remodeling remains to be determined. We investigated the effects of 8 weeks of paternal resistance training (RT) on offspring tendon proteome exposed to standard diet or HF diet. Wistar rats were randomly divided into two groups: sedentary fathers and trained fathers (8 weeks, three times per week, with 8–12 dynamic movements per climb in a stair climbing apparatus). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into four groups (five animals per group): offspring from sedentary fathers were exposed either to control diet (SFO-C), or to high-fat diet (SFO-HF); offspring from trained fathers were exposed to control diet (TFO-C) or to a high-fat diet (TFO-HF). The Nano-LC-MS/MS analysis revealed 383 regulated proteins among offspring groups. HF diet induced a decrease of abundance in tendon proteins related to extracellular matrix organization, transport, immune response and translation. On the other hand, the changes in the offspring tendon proteome in response to paternal RT were more pronounced when the offspring were exposed to HF diet, resulting in positive regulation of proteins essential for the maintenance of tendon integrity. Most of the modulated proteins are associated to biological pathways related to tendon protection and damage recovery, such as extracellular matrix organization and transport. The present study demonstrated that the father’s lifestyle could be crucial for tendon homeostasis in the first generation. Our results provide important insights into the molecular mechanisms involved in paternal intergenerational effects and potential protective outcomes of paternal RT