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Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-ß1) were investigated. Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-ß1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the stimulation protocol for the optimal chondrogenic differentiation of synovial explants.

The inner surface layer of human joints, the synovium, is a source of stem cells for the repair of articular cartilage defects. We investigated the potential of the normal human synovium to form novel cartilage and compared its chondrogenic capacity with that of two patient groups suffering from major joint diseases: young adults with femoro-acetabular impingement syndromes of the hip (FAI), and elderly individuals with osteoarthritic degeneration of the knee (OA). Synovial membrane explants of these three patient groups were induced in vitro to undergo chondrogenesis by growth factors: bone morphogenetic protein-2 (BMP-2) alone, transforming growth factor-β1 (TGF-β1) alone, or a combination of these two. Quantitative evaluations of the newly formed cartilages were performed respecting their gene activities, as well as the histochemical, immunhistochemical, morphological and histomorphometrical characteristics. Formation of adult articular-like cartilage was induced by the BMP-2/TGF-β1 combination within all three groups, and was confirmed by adequate gene-expression levels of the anabolic chondrogenic markers; the levels of the catabolic markers remained low. Our data reveal that the chondrogenic potential of the normal human synovium remains uncompromised, both in FAI and OA. The potential of synovium-based clinical repair of joint cartilage may thus not be impaired by age-related joint pathologies.

Miniscrew implants (MSIs) have been widely used as temporary anchorage devices in orthodontic clinics. However, one of their major limitations is the relatively high failure rate. We hypothesize that a biomimetic calcium phosphate (BioCaP) coating layer on mini-pin implants might be able to accelerate the osseointegration, and can be a carrier for biological agents. A novel mini-pin implant to mimic the MSIs was used. BioCaP (amorphous or crystalline) coatings with or without the presence of bovine serum albumin (BSA) were applied on such implants and inserted in the metaphyseal tibia in rats. The percentage of bone to implant contact (BIC) in histomorphometric analysis was used to evaluate the osteoconductivity of such implants from six different groups (n=6 rats per group): (1) no coating no BSA group, (2) no coating BSA adsorption group, (3) amorphous BioCaP coating group, (4) amorphous BioCaP coating-incorporated BSA group, (5) crystalline BioCaP coating group, and (6) crystalline BioCaP coating-incorporated BSA group. Samples were retrieved 3 days, 1 week, 2 weeks, and 4 weeks post-surgery. The results showed that the crystalline BioCaP coating served as a drug carrier with a sustained release profile. Furthermore, the significant increase in BIC occurred at week 1 in the crystalline coating group, but at week 2 or week 4 in other groups. These findings indicate that the crystalline BioCaP coating can be a promising surface modification to facilitate early osseointegration and increase the success rate of miniscrew implants in orthodontic clinics.

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded alpha1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

The interaction of chondrocytes with the extracellular‐matrix environment is mediated mainly by integrins. Ligated integrins are recruited to focal adhesions (FAs) together with scaffolding proteins and kinases, such as integrin‐linked kinase (Ilk). Ilk binds the cytoplasmic domain of β1‐, β2‐ and β3‐integrins and recruits adaptors and kinases, and is thought to stimulate downstream signalling events through phosphorylation of protein kinase B/Akt (Pkb/Akt) and glycogen synthase kinase 3‐β (GSK3‐β). Here, we show that mice with a chondrocyte‐specific disruption of the gene encoding Ilk develop chondrodysplasia, and die at birth due to respiratory distress. The chondrodysplasia was characterized by abnormal chondrocyte shape and decreased chondrocyte proliferation. In addition, Ilk‐deficient chondrocytes showed adhesion defects, failed to spread and formed fewer FAs and actin stress fibres. Surprisingly, phosphorylation of Pkb/Akt and GSK3‐β is unaffected in Ilk‐deficient chondrocytes. These findings suggest that Ilk regulates actin reorganization in chondrocytes and modulates chondrocyte growth independently of phosphorylation of Pkb/Akt and GSK3‐β.

A number of arthroscopic procedures that are used in the treatment of focal cartilage lesions or osteoarthritic joints, such as shaving, débridement, and laser abrasion, involve the removal of both diseased and healthy articular cartilage. The excision of such tissue has the effect of generating lesions within the articular cartilage. The fate of the chondrocytes that border such lesions has not been evaluated. The purpose of this investigation was to ascertain whether the surgical creation of lesions in articular cartilage induces irreversible loss of chondrocytes over time from tissue bordering the wound edge and to determine whether the synthetic activity of cells in this region is compromised. Partial-thickness defects of defined dimensions were created in the femoral condyle and/or trochlear groove of rabbits and miniature pigs. Cell volumes, cell volume densities, and numerical cell densities within tissue close to (within 100 micro m) and remote from (control site) the wound edge were determined by quantitative histomorphometry at various time intervals up to six months after surgery. Rates of proteoglycan synthesis by cells in both regions were determined by quantitative autoradiography following (35) S-sulphate labeling in vivo. The surgical creation of partial-thickness lesions in articular cartilage induced a significant and long-term loss of cells from tissue near the wound edge. However, the surviving cell population maintained a normal rate of matrix proteoglycan deposition. This study illustrates that maintenance and remodeling of cartilage matrix close to wound edges in articular cartilage lesions is compromised, since fewer cells, with an unchanged metabolic activity rate, are left to sustain matrix domains.

This preliminary study investigates the differences between experimental periodontitis and peri-implantitis in a dog model, with a focus on the histopathology, inflammatory responses, and specific immunoregulatory activities driven by Th1/Th2-positive cells. Twelve dental implants were inserted into the edentulated posterior mandibles of 6 beagle dogs and were given 12 weeks for osseointegration. Experimental peri-implantitis and periodontitis (first mandible molar) were then induced using cotton-floss ligatures. Twelve weeks later, alveolar bones were quantitated by cone beam-computer tomography. Histopathologic analysis of the inflamed gingiva and periodontal tissues was performed by light microscopy, and the Th1/Th2 cell populations were investigated by flow cytometry. Peri-implantitis and periodontitis were both found to be associated with pronounced bone resorption effects, both to a similar degree vertically, but with a differential bone resorption pattern mesio-distally, and with a significantly higher and consistent bone resorption result in peri-implantitis, although with a higher variance of bone resorption in periodontitis. The histologic appearances of the inflammatory tissues were identical. The percentages of Th1/Th2 cells in the inflamed gingival tissues of both experimental peri-implantitis and periodontitis were also found to be similar. Experimental periodontitis and peri-implantitis in the dog model show essentially the same cellular pathology of inflammation. However, bone resorption was found to be significantly higher in peri-implantitis; the histopathologic changes in the periodontal tissues were similar in both groups but showed a higher interindividual variation in periodontitis and appeared more uniform in peri-implantitis. This preliminary study indicates that more focused experimental in vivo inflammation models need to be developed to better simulate the human pathology in the 2 different diseases and to have a valuable tool to investigate more specifically how novel treatments/prevention approaches may heal the differential adverse effects on bone tissue and on periodontium in periodontitis and in periimplantitis.

Absorbed collagen sponge (ACS)/bone morphogenetic protein-2 (BMP-2) are widely used in clinical practise for bone regeneration. However, the application of this product was found to be associated with a significant pro-inflammatory response, particularly in the early phase after implantation. This study aimed to clarify if the pro-inflammatory activities, associated with BMP-2 added to ACS, were related to the physical state of the carrier itself, i.e., a wet or a highly dehydrated state of the ACS, to the local degree of vascularisation and/or to local biomechanical factors. ACS (0.8 cm diameter)/BMP-2 were implanted subcutaneously in the back of 12 eight-week-old Sprague Dawley rats. Two days after surgery, the implanted materials were retrieved and analysed histologically and histomorphometrically. The acute inflammatory response following implantation of ACS was dependent of neither the presence or absence of BMP-2 nor the degree of vascularization in the surrounding tissue nor the hydration state (wet versus dry) of the ACS material at the time of implantation. Differential micro biomechanical factors operating at the implantation site appeared to have an influence on the thickness of inflammation. We conclude that the degree of the early inflammatory response of the ACS/BMP-2 may be associated with the physical and chemical properties of the carrier material itself.

(1) Background: We tested the hypothesis that hyaluronic acid (HA) can significantly promote the osteogenic potential of BMP-2/ACS (absorbable collagen sponge), an efficacious product to heal large oral bone defects, thereby allowing its use at lower dosages and, thus, reducing its side-effects due to the unphysiologically-high doses of BMP-2; (2) Methods: In a subcutaneous bone induction model in rats, we first sorted out the optimal HA-polymer size and concentration with micro CT. Thereafter, we histomorphometrically quantified the effect of HA on new bone formation, total construct volume, and densities of blood vessels and macrophages in ACS with 5, 10, and 20 μg of BMP-2; (3) Results: The screening experiments revealed that the 100 µg/mL HA polymer of 48 kDa molecular weight could yield the highest new bone formation. Eighteen days post-surgery, HA could significantly enhance the total volume of newly-formed bone by approximately 100%, and also the total construct volume in the 10 μg BMP-2 group. HA could also significantly enhance the numerical area density of blood vessels in 5 μg BMP-2 and 10 μg BMP-2 groups. HA did not influence the numerical density of macrophages; and (4) Conclusions: An optimal combined administration of HA could significantly promote osteogenic and angiogenic activity of BMP-2/ACS, thus potentially minimizing its potential side-effects.

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2 −/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.