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Enhancement of cartilage repair through the addition of growth plate chondrocytes in an immature skeleton animal model.

ABSTRACT: BACKGROUND:The treatment of articular cartilage damage is a major clinical problem. More often, this clinical issue affects children, which forces doctors to find the best treatment method. METHODS:The aim of this experimental study on 2-month-old Landrace pigs was to compare the results of two cartilage defect treatments: (1) filling the cartilage defect with a scaffold incubated with bone marrow aspirate supplemented with growth plate chondrocytes (the CELLS group) and (2) filling the cartilage defect with an empty scaffold implanted after drilling the subchondral bone (the CTRL group). The treatment outcomes were assessed macroscopically and microscopically. RESULTS:Based on the macroscopic evaluation, all animals showed a nearly normal morphology, with an average of 9.66/12 points (CTRL) and 10.44/12 points (CELLS). Based on the microscopic evaluation, 1 very good result and 8 good results were obtained in the CTRL group, with an average of 70.44%, while 5 very good results and 4 good results were obtained in the CELLS group, with an average of 79.61%. CONCLUSIONS:(1) Growth plate chondrocytes have high chondrogenic potential and thus offer new possibilities for cartilage cell therapy. (2) The implantation of a scaffold loaded with bone marrow-derived MSCs (mesenchymal stem cells) and growth plate chondrocytes into a cartilage defect is a good therapeutic method in immature patients. (3) Cartilage repair based on a scaffold with bone marrow aspirate-derived cells supplemented with autologous growth plate chondrocytes achieves better results than repair with marrow stimulation and a hyaluronic acid-based scaffold (overall microscopic rating). (4) Chondrocyte clustering is a manifestation of the cartilage repair process but requires further observation.

SUBMITTER: Tomaszewski R

PROVIDER: S-EPMC6694631 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Enhancement of cartilage repair through the addition of growth plate chondrocytes in an immature skeleton animal model.

Tomaszewski Ryszard R Wiktor Łukasz Ł Gap Artur A

Journal of orthopaedic surgery and research 20190815 1

<h4>Background</h4>The treatment of articular cartilage damage is a major clinical problem. More often, this clinical issue affects children, which forces doctors to find the best treatment method.<h4>Methods</h4>The aim of this experimental study on 2-month-old Landrace pigs was to compare the results of two cartilage defect treatments: (1) filling the cartilage defect with a scaffold incubated with bone marrow aspirate supplemented with growth plate chondrocytes (the CELLS group) and (2) filli ...[more]

PMID: 31416470

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Project description:Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.

Project description:We previously found that FoxA factors are necessary for chondrocyte differentiation. To investigate whether FoxA factors alone are sufficient to drive chondrocyte hypertrophy, we build a FoxA2 transgenic mouse in which FoxA2 cDNA is driven by a reiterated Tetracycline Response Element (TRE) and a minimal CMV promoter. This transgenic line was crossed with a col2CRE;Rosa26rtTA/+ mouse line to generate col2CRE;Rosa26rtTA/+;TgFoxA2+/- mice for inducible expression of FoxA2 in cartilage using doxycycline treatment. Ectopic expression of FoxA2 in the developing skeleton reveals skeletal defects and shorter skeletal elements in E17.5 mice. The chondro-osseous border was frequently mis-shaped in mutant mice, with small islands of col.10+ hypertrophic cells extending in the metaphyseal bone. Even though overexpression of FoxA2 causes an accumulation of hypertrophic chondrocytes, it did not trigger ectopic hypertrophy in the immature chondrocytes. This suggests that FoxA2 may need transcriptional co-factors (such as Runx2), whose expression is restricted to the hypertrophic zone, and absent in the immature chondrocytes. To investigate a potential FoxA2/Runx2 interaction in immature chondrocytes versus hypertrophic cells, we separated these two subpopulations by FACS to obtain CD24+CD200+ hypertrophic chondrocytes and CD24+CD200- immature chondrocytes and we ectopically expressed FoxA2 alone or in combination with Runx2 via lentiviral gene delivery. In CD24+CD200+ hypertrophic chondrocytes, FoxA2 enhanced the expression of chondrocyte hypertrophic markers collagen 10, MMP13, and alkaline phosphatase. In contrast, in the CD24+CD200- immature chondrocytes, neither FoxA2 nor Runx2 overexpression could induce ectopic expression of hypertrophic markers MMP13, alkaline phosphatase, or PTH/PTHrP receptor. Overall these findings mirror our in vivo data, and suggest that induction of chondrocyte hypertrophy by FoxA2 may require other factors in addition to Runx2 (i.e., Hif2α, MEF2C, or perhaps unknown factors), whose expression/activity is rate-limiting in immature chondrocytes.

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Enhancement of cartilage repair through the addition of growth plate chondrocytes in an immature skeleton animal model.

Publications

Enhancement of cartilage repair through the addition of growth plate chondrocytes in an immature skeleton animal model.

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