The role of the microenvironment in growth plate and articular chondrocyte differentiation

Abstract: One crucial process during skeletal development is joint formation. It starts with the interzone appearance characterized by high expression of Gdf5, Gli3, Wnt4 and Wnt9a, low expression of Col2a1 of the local compact elongated cells which subsequently give rise to most joint elements including articular cartilage as the ongoing joint cavitation. PRG4 cannot be detected in superficial chondrocytes covering the joint surface until the cavitation is complete. Lineage tracing study has shown that these flattened Prg4 + chondrocytes play the role of progenitors giving rise to the deeper-layer chondrocytes. The joint cavity is filled up by lubricant synovial fluid the blood-ultrafiltrate of which contains abundant hyaluronic acid, and together with the action of lubricin, the friction at the joint surface is minimized. Growth plate cartilage has a similar layer characterized structure yet distinctive function and fate from articular cartilage. Longitudinal bone growth is realized by a stepwise chondrocyte proliferation and differentiation program in the growth plate. Slowly dividing resting zone chondrocytes differentiate into the underlying proliferative chondrocytes that undergo clonal expansion and orient along the long axis of the bone, forming columns of cells. Post-proliferative chondrocytes undergo hypertrophic differentiation, mineralize the matrix, attract invading vessels and bone cells and are replaced by bone. In spite of a common origin, articular and growth plate chondrocytes are believed belonging to different lineages. But the related cellular and molecular mechanism responsible for such a divergence has not been clear. Though several clinical articular cartilage repair practices such as autologous chondrocyte implantation and allograft transplantation have been continuously refined, the lack of a clear mechanistic landscape about articular cartilage homeostasis and knowledge of the actual contribution of transplanted cells always lead to variant outcomes. In study I, we first systematically tested and optimized critical steps of the techniques required to assess chondrocyte differentiation in the subsequent studies. For skeletal tissues, proteolytic-induced epitope retrieval (PIER) was superior to heat-induced epitope retrieval (HIER) as it unmasks the antigens with preserved morphology and it was useful for in situ hybridization (ISH) as well as for immunohistochemistry (IHC) and immunofluorescence (IF). However, experimental parameters including enzyme concentration, its incubation time, temperature and time of DNA hydrolysis, must be carefully optimized to ensure high assay sensitivity and specificity. In study II, with the optimized toolbox above, we tested our hypothesis that the synovial microenvironment inhibits chondrocyte hypertrophy and promotes articular cartilage differentiation. We ectopically transplanted growth plate into the distal femur surface in recipient animals and found that no hypertrophic changes occurred in the transplanted growth plate cartilage anymore at the joint surface, instead, an articular like cartilage was formed. We then used the in vitro model of cell pellet cultures with the synoviocyte conditioned or the control chondrogenic media to validate the in vivo findings. Likewise, it was found that the expression of hypertrophic differentiation markers was down regulated whereas articular surface marker Prg4 was effectively induced in pellets exposed to the conditioned media. Further exploratory studies suggested that the synoviocyte conditioned media factor is a large-sized (> 35kDa) and heat resistant protein mainly secreted by synoviocytes. In conclusion, we found a novel mechanism by which the local synovial microenvironment suppresses chondrocyte hypertrophy at the articular surface and promote articular cartilage differentiation. In study III, we hypothesized whether the prenatally programmed fate and function of epiphyseal and articular chondrocytes can be influenced by postnatal local cellular microenvironment. We transplanted the donor articular-epiphyseal cartilage complex (AECC) autografts in inverted orientation to recipient sites with different weight-bearing properties in goats. Macroscopic, histological and differentiation status of grafts were followed up by the end of postoperative week 1, 2, 3, 6, 12, and 24. It was found that grafts localized at the trochlea and lateral condyle were with more cartilaginous appearance through 6 weeks post-surgery compared to the medial condyle, and epiphyseal chondrocytes transplanted onto the articular surface differentiate into superficial articular like chondrocytes which are densely packed with smaller and flattened morphology. In conclusion, these findings suggested that the synovial local microenvironment may contain biochemical and or biomechanical factors promoting articular cartilage differentiation while inhibiting endochondral ossification, despite the articular and epiphyseal growth chondrocytes displayed resilience to postnatal transdifferentiation within the surgically switched local cellular environment. In study IV, the role of the transplanted perichondrial cells in healing the resurfaced joint surface was investigated. We harvested perichondrium and periosteum allografts from the eGFP + donor rats and ectopically transplanted them to the full thickness articular cartilage defect at the distal femur in wild-type littermates. Surgery femurs were collected at postoperative day 3, 14, 56, and 112 for transplanted cells tracing, microscopic, histological and differentiation analysis. It was shown that almost all cells at the defect are transplant derived. Perichondrium initially has abundant SOX9 + cells that with time differentiate into the expanding hyaline cartilage positive to Col2a1 and negative to Col1a1, and the proteoglycan rich matrix at the injury sites. Notably, at latter time points, the perichondrium derived cartilage actively remodeled into bone at its borderline next to the underlying subchondral bone, and at day 112 post-surgery Prg4 expression was detected in perichondrium derived superficial chondrocytes. Periosteum initially lack SOX9 expression which later underwent a transient increase however, with time it formed fibrous layer with thinning thickness, and it provided the derived cells into the subchondral bone. To conclude, the transplanted perichondrium and periosteum transplants did not only stimulate regeneration responses but also by themselves transformed into the regenerated tissue. Perichondrium grafts differentiated into hyaline cartilaginous structure resembling articular cartilage, whereas periosteum gradually formed a thinning fibrous layer. In summary, this project systematically optimized skeletal tissue-based targeting assays, provided evidence of protective factors existing in the synovial microenvironment and explored the potential properties of this factor. Therefore, it sheds light on a novel mechanistic signaling transduced by the interaction between the bioactive synovial factor(s) and articular cartilage superficial chondrocytes which are protected from hypertrophy and ossification. Besides, a reference was given for the chondrogenesis of perichondrium and periosteum transplants when used to reconstruct the full thickness defected articular cartilage.

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