Ex vivo organotypic bone slice culture reveals preferential chondrogenesis after sustained growth plate injury.

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15-30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 µm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption. In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine.

Proton and Carbon Ion Irradiation Changes the Process of Endochondral Ossification in an Ex Vivo Femur Organotypic Culture Model.

Particle therapy (PT) that utilizes protons and carbon ions offers a promising way to reduce the side effects of radiation oncology, especially in pediatric patients. To investigate the influence of PT on growing bone, we exposed an organotypic rat ex vivo femur culture model to PT. After irradiation, histological staining, immunohistochemical staining, and gene expression analysis were conducted following 1 or 14 days of in vitro culture (DIV). Our data indicated a significant loss of proliferating chondrocytes at 1 DIV, which was followed by regeneration attempts through chondrocytic cluster formation at 14 DIV. Accelerated levels of mineralization were observed, which correlated with increased proteoglycan production and secretion into the pericellular matrix. Col2α1 expression, which increased during the cultivation period, was significantly inhibited by PT. Additionally, the decrease in ColX expression over time was more pronounced compared to the non-IR control. The chondrogenic markers BMP2, RUNX2, OPG, and the osteogenic marker ALPL, showed a significant reduction in the increase in expression after 14 DIV due to PT treatment. It was noted that carbon ions had a stronger influence than protons. Our bone model demonstrated the occurrence of pathological and regenerative processes induced by PT, thus building on the current understanding of the biological mechanisms of bone.

Disruption of Endochondral Ossification and Extracellular Matrix Maturation in an Ex Vivo Rat Femur Organotypic Slice Model Due to Growth Plate Injury.

Postnatal bone fractures of the growth plate (GP) are often associated with regenerative complications such as growth impairment. In order to understand the underlying processes of trauma-associated growth impairment within postnatal bone, an ex vivo rat femur slice model was developed. To achieve this, a 2 mm horizontal cut was made through the GP of rat femur prior to the organotypic culture being cultivated for 15 days in vitro. Histological analysis showed disrupted endochondral ossification, including disordered architecture, increased chondrocyte metabolic activity, and a loss of hypertrophic zone throughout the distal femur. Furthermore, altered expression patterns of Col2α1, Acan, and ColX, and increased chondrocyte metabolic activity in the TZ and MZ at day 7 and day 15 postinjury were observed. STEM revealed the presence of stem cells, fibroblasts, and chondrocytes within the injury site at day 7. In summary, the findings of this study suggest that the ex vivo organotypic GP injury model could be a valuable tool for investigating the underlying mechanisms of GP regeneration post-trauma, as well as other tissue engineering and disease studies.

Inflammatory Pre-Conditioning of Adipose-Derived Stem Cells with Cerebrospinal Fluid from Traumatic Brain Injury Patients Alters the Immunomodulatory Potential of ADSC Secretomes.

Immunomodulation by adipose-tissue-derived stem cells (ADSCs) is of special interest for the alleviation of damaging inflammatory responses in central nervous system injuries. The present study explored the effects of cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients on this immunomodulatory potential of ADSCs. CSF conditioning of ADSCs increased messenger RNA levels of both pro- and anti-inflammatory genes compared to controls. Exposure of phorbol-12-myristate-13-acetate-differentiated THP1 macrophages to the secretome of CSF-conditioned ADSCs downregulated both proinflammatory (cyclooxygenase-2, tumor necrosis factor alpha) and anti-inflammatory (suppressor of cytokine signaling 3, interleukin-1 receptor antagonist, and transforming growth factor beta) genes in these cells. Interleukin-10 expression was elevated in both naïve and conditioned secretomes. ADSC secretome treatment, further, induced macrophage maturation of THP1 cells and increased the percentage of CD11b+, CD14+, CD86+, and, to a lesser extent, CD206+ cells. This, moreover, enhanced the phagocytic activity of CD14+ and CD86+ cells, though independently of pre-conditioning. Secretome exposure, finally, also induced a reduction in the percentage of CD192+ adherent cells in cultures of peripheral blood mononuclear cells (PBMCs) from both healthy subjects and TBI patients. This limited efficacy (of both naïve and pre-conditioned secretomes) suggests that the effects of lymphocyte-monocyte paracrine signaling on the fate of cultured PBMCs are strongest upon adherent cell populations.