In major joint diseases the human synovium retains its potential to form repair cartilage.

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-ß1 (TGF-ß1) 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-ß1 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.

Human Bone Typing Using Quantitative Cone-Beam Computed Tomography.

INTRODUCTION: Bone typing is crucial to enable the choice of a suitable implant, the surgical technique, and the evaluation of the clinical outcome. Currently, bone typing is assessed subjectively by the surgeon. OBJECTIVE: The aim of this study is to establish an automatic quantification method to determine local bone types by the use of cone-beam computed tomography (CBCT) for an observer-independent approach. METHODS: Six adult human cadaver skulls were used. The 4 generally used bone types in dental implantology and orthodontics were identified, and specific Hounsfield unit (HU) ranges (grey-scale values) were assigned to each bone type for identification by quantitative CBCT (qCBCT). The selected scanned planes were labelled by nonradiolucent markers for reidentification in the backup/cross-check evaluation methods. The selected planes were then physically removed as thick bone tissue sections for in vitro correlation measurements by qCBCT, quantitative micro-computed tomography (micro-CT), and quantitative histomorphometry. RESULTS: Correlation analyses between the different bone tissue quantification methods to identify bone types based on numerical ranges of HU values revealed that the Pearson correlation coefficient of qCBCT with micro-CT and quantitative histomorphometry was R = 0.9 (P = .001) for all 4 bone types . CONCLUSIONS: We found that  qCBCT can reproducibly and objectively assess human bone types at implant sites.

Crystalline Biomimetic Calcium Phosphate Coating on Mini-Pin Implants to Accelerate Osseointegration and Extend Drug Release Duration for an Orthodontic Application.

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.

The Synovium of Human Osteoarthritic Joints Retains Its Chondrogenic Potential Irrespective of Age.

The autologous synovium is a potential tissue source for local induction of chondrogenesis by tissue engineering approaches to repair articular cartilage defects that occur in osteoarthritis. It was the aim of the present study to ascertain whether the aging of human osteoarthritic patients compromises the chondrogenic potential of their knee-joint synovium and the structural and metabolic stability of the transformed tissue. The patients were allocated to one of the following two age categories: 54-65 years and 66-86 years (n = 7-11 donors per time point and experimental group; total number of donors: 64). Synovial biopsies were induced in vitro to undergo chondrogenesis by exposure to bone morphogenetic protein-2 (BMP-2) alone, transforming growth factor-ß1 (TGF-ß1) alone, or a combination of the two growth factors, for up to 6 weeks. The differentiated explants were evaluated morphologically and morphometrically for the volume fraction of metachromasia (sulfated proteoglycans), immunohistochemically for type-II collagen, and for the gene expression levels of anabolic chondrogenic markers as well as catabolic factors by a real-time polymerase chain reaction analysis. Quantitative metachromasia revealed that chondrogenic differentiation of human synovial explants was induced to the greatest degree by either BMP-2 alone or the BMP-2/TGF-ß1 combination, that is, to a comparable level with each of the two stimulation protocols and within both age categories. The BMP-2/TGF-ß1combination protocol resulted in chondrocytes of a physiological size for normal human articular cartilage, unlike the BMP-2-alone stimulation that resulted in cell sizes of terminal hypertrophy. The stable gene expression levels of the anabolic chondrogenic markers confirmed the superiority of these two stimulation protocols and demonstrated the hyaline-like qualities of the generated cartilage matrix. The gene expression levels of the catabolic markers remained extremely low. The data also confirmed the usefulness of experimental in vitro studies with bovine synovial tissue as a paradigm for human synovial investigations. Our data reveal the chondrogenic potential of the human knee-joint synovium of osteoarthritic patients to be uncompromised by aging and catabolic processes. The potential of synovium-based clinical engineering (repair) of cartilage tissue using autologous synovium may thus not be reduced by the age of the human patient. Impact statement Our data reveal that in younger and older age groups alike, synovial explants from osteoarthritic joints can be equally well induced to undergo chondrogenesis in vitro; that is, the chondrogenic potential of the human synovium is not compromised by aging. These findings imply that the autologous synovium represents an adequate tissue source for the repair of articular cartilage in clinical practice by tissue engineering approaches in human patients suffering from osteoarthritis, independent of the patient's age.

Anticytokine Activity Enhances Osteogenesis of Bioactive Implants.

In dental clinical practice, systemic steroids are often applied at the end of implant surgeries to reduce postsurgical inflammation (tissue swelling, etc.) and to reduce patient discomfort. However, the use of systemic steroids is associated with generalized catabolic effects and with a temporarily reduced immunological competence. We hypothesize that by applying locally anticytokine antibodies (antitumor necrosis factor alpha and anti-interleukin-1 beta) together with a bioactive osteogenic implant at the time of the surgical intervention for the placement of a construct, we will be able to achieve the same beneficial effects as those using systemic steroids but are able to avoid the generalized antianabolic effects and the reduced immunocompetence effects, associated with the systemic use of steroids. In an adult rat model, a collagen sponge, soaked with the osteogenic agent bone morphogenetic protein-2, was used as an example for a bioactive implant material and was surgically placed subcutaneously. In the acute inflammatory phase after implantation (2 days after surgery) we investigated the local inflammatory tissue response, and 18 days postsurgically the efficiency of local osteogenesis (to assess possible antianabolic effects). We found that the negative control groups, treated postsurgically with systemic steroids, showed a significant suppression of both the inflammatory response and the osteogenetic activity, that is, they were associated with significant general antianabolic effects, even when steroids were used only at a low dose level. The local anticytokine treatment, however, was able to significantly enhance new bone formation activity, that is, the anabolic activity, over positive control values with BMP-2 only. However, the anticytokine treatment was unable to reduce the local inflammatory and swelling responses.

Induced Experimental Peri-Implantitis and Periodontitis: What Are the Differences in the Inflammatory Response?

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.

Polymeric mesh and insulin-like growth factor 1 delivery enhance cell homing and graft-cartilage integration.

Cartilage injury, such as full-thickness lesions, predisposes patients to the premature development of osteoarthritis, a degenerative joint disease. While surgical management of cartilage lesions has improved, long-term clinical efficacy has stagnated, owing to the lack of hyaline cartilage regeneration and inadequate graft-host integration. This study tests the hypothesis that integration of cartilage grafts with native cartilage can be improved by enhancing the migration of chondrocytes across the graft-host interface via the release of chemotactic factor from a degradable polymeric mesh. To this end, a polylactide-co-glycolide/poly-ε-caprolactone mesh was designed to localize the delivery of insulin-like growth factor 1 (IGF-1), a well-established chondrocyte attractant. The release of IGF-1 (100 ng/mg) enhanced cell migration from cartilage explants, and the mesh served as critical structural support for cell adhesion, growth, and production of a cartilaginous matrix in vitro, which resulted in increased integration strength compared with mesh-free repair. Further, this neocartilage matrix was structurally contiguous with native and grafted cartilage when tested in an osteochondral explant model in vivo. These results demonstrate that this combined approach of a cell homing factor and supportive matrix will promote cell-mediated integrative cartilage repair and improve clinical outcomes of cartilage grafts in the treatment of osteoarthritis.

Enhanced biocompatibility and improved osteogenesis of coralline hydroxyapatite modified by bone morphogenetic protein 2 incorporated into a biomimetic coating.

OBJECTIVES: (1) To determine whether the biocompatibility of coralline hydroxyapatite (CHA) granules could be improved by using an octacalcium phosphate (OCP) coating layer, and/or functionalized with bone morphogenetic protein 2 (BMP-2), and (2) to investigate if BMP-2 incorporated into this coating is able to enhance its osteoinductive efficiency, in comparison to its surface-adsorbed delivery mode. METHODS: CHA granules (0.25 g per sample) bearing a coating-incorporated depot of BMP-2 (20 µg/sample) together with the controls (CHA bearing an adsorbed depot of BMP-2; CHA granules with an OCP coating without BMP-2; pure CHA granules) were implanted subcutaneously in rats (n = 6 animals per group). Five weeks later, the implants were retrieved for histomorphometric analysis to quantify the volume of newly generated bone, bone marrow, fibrous tissue and foreign body giant cells (FBGCs). The osteoinductive efficiency of BMP-2 and the rates of CHA degradation were also determined. RESULTS: The group with an OCP coating-incorporated depot of BMP-2 showed the highest volume and quality or bone, and the highest osteoinductive efficacy. OCP coating was able to reduce inflammatory responses (improve biocompatibility), and also simple adsorption of BMP-2 to CHA achieved this. CONCLUSIONS: The biocompatibility of CHA granules (reduction of inflammation) was significantly improved by coating with a layer of OCP. Pure surface adsorption of BMP-2 to CHA also reduced inflammation. Incorporation of BMP-2 into the OCP coatings was associated with the highest volume and quality of bone, and the highest biocompatibility degree of the CHA granules. CLINICAL SIGNIFICANCE: Higher osteoinductivity and improved biocompatibility of CHA can be obtained when a layer of BMP-2 functionalized OCP is deposited on the surfaces of CHA granules.

Cells, soluble factors and matrix harmonically play the concert of allograft integration.

Implantation of allograft tissues has massively grown over the last years, especially in the fields related to sports medicine. Beside the fact that often no autograft option exists, autograft related disadvantages as donor-site morbidity and prolonged operative time are drastically reduced with allograft tissues. Despite the well documented clinical success for bone allograft procedures, advances in tissue engineering raised the interest in meniscus, osteochondral and ligament/tendon allografts. Notably, their overall success rates are constantly higher than 80%, making them a valuable treatment option in orthopaedics, especially in knee surgery. Complications reported for allografting procedures are a small risk of disease transmission, immunologic rejection, and decreased biologic incorporation together with nonunion at the graft-host juncture and, rarely, massive allograft resorption. Although allografting is a successful procedure, improved techniques and biological knowledge to limit these pitfalls and maximize graft incorporation are needed. A basic understanding of the biologic processes that affect the donor-host interactions and eventual incorporation and remodelling of various allograft tissues is a fundamental prerequisite for their successful clinical use. Further, the importance of the interaction of immunologic factors with the biologic processes involved in allograft incorporation has yet to be fully dissected. Finally, new tissue engineering techniques and use of adjunctive growth factors, cell based and focused gene therapies may improve the quality and uniformity of clinical outcomes. The aim of this review is to shed light on the biology of meniscus, osteochondral and ligament/tendon allograft incorporation and how collection and storage techniques may affect graft stability and embodiment.Level of evidence V.

A Novel Experimental Dental Implant Permits Quantitative Grading of Surface-Property Effects on Osseointegration.

PURPOSE: To test the hypothesis if a novel single-chamber experimental dental implant allows in vivo the quantitative assessment of osseointegration over time and as a function of different surface properties (physical, chemical, geometric, biologic [osteoconductive or osteoinductive]) in a biologically unfavorable environment (local osteoporosis). MATERIALS AND METHODS: Three prototypes of a novel experimental implant with different chamber sizes (small, medium, and large) were compared with each other to find out the minimum size of bone chambers needed to allow a discriminative quantification of osseointegration over time. For the comparison of low and high surface osteoconductivity properties, conventional sandblasted, acid-etched chamber surfaces (low surface osteoconductivity) were compared with biomimetically (calcium phosphate) coated ones (high surface osteoconductivity). The implants (4 implants per animal; 88 implants per time point) were inserted into the edentulous maxillae of a total of 66 adult goats with a physiologically osteoporotic masticatory apparatus. Two, 4, and 8 weeks later, they were excised and prepared for a histomorphometric analysis of the volume of neoformed bone within the chamber space and of the bone-to-implant contact (BIC) area. RESULTS: The implants with small chambers did not show significant differences in bone coverage (BIC) nor bone volume (relative and absolute volume), neither as a function of time nor as of implant surface property (low versus high surface osteoconductivity). However, medium and large chambers revealed significant differences respecting both of these parameters over the 8-week postoperative time period. CONCLUSION: The new implant model permits a discriminative quantification of osseointegration in vivo in an osteoporotic bone environment for implants with medium-sized and large-sized chambers. Quantitative assessment of osseointegration is possible, both over time and as a function of low and high surface osteoconductivity properties.

Elongation of the Long Bones in Humans by the Growth Plates.

The disk of hyaline cartilage that is interposed between the epiphysis and the metaphysis of each of the long bones is responsible for its elongation, and, thus, when the lower limbs are concerned, for increases in bodily height. This so-called growth plate is avascular, aneural, and alymphatic. It consists solely of chondrocytes and an extracellular matrix which the cells elaborate. The growth plate is architectonically striking in so far as the chondrocytes are aligned in strictly vertical columns, which represent the functional units of longitudinal bone growth. The growth process begins with the slow division of chondrocytes in the resting ("stem cell") zone and proceeds with their rapid proliferation in the adjacent zone. These cells then undergo a process of progressive enlargement, which culminates in the zone of terminal hypertrophy. The life history of any given cell is recapitulated in a vertical column. The neoformation of cartilage in the axial direction is synchronized with its destruction at the vascular invasion front of the metaphysis and results in an elongation of the bony trabeculae. The mechanism that governs the highly coordinated sequence of events that underlies the growth of the long bones is complex; it is subject to influence by genetic, hormonal, nutritional, environmental, and pathological factors.

The kinetics and mechanism of bone morphogenetic protein 2 release from calcium phosphate-based implant-coatings.

Biomimetically deposited calcium phosphate-based coatings of prostheses can serve as a vehicle for the targeted delivery of growth factors to the local implant environment. Based on indirect evidence in previous studies we hypothesize that such agents are liberated gradually from the coating via a cell-mediated degradation. In the present study, we tested this hypothesis by investigating the release mechanism and its kinetics by use of a radiolabeled osteogenic agent (131 I-BMP-2) under conditions in which native cell populations with a coating-degradative potential were either absent or present. The release of 131 I-BMP-2 was monitored for 5 weeks, either in vitro or after implantation at an ectopic (subcutaneous) site in rats in vivo. Only from implants that bore a coating-incorporated depot of bone morphogenetic protein 2 (BMP-2) was the agent released slowly and steadily over 5 weeks, that is, 50% of the loaded dose was liberated in vivo (5 to 10% weekly), as against 14.6% in vitro (less than 1% weekly). The coatings bearing an incorporated depot of BMP-2 underwent significant cell-mediated degradation, whereas under cell-free conditions no degradation occurred, and the spontaneous release of BMP-2 was negligible. Our findings confirm this carrier system to be a suitable vehicle for the sustained and cell-mediated delivery of BMP-2. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2363-2371, 2018.

The Acute Inflammatory Response to Absorbed Collagen Sponge Is Not Enhanced by BMP-2.

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.

Hyaluronic Acid Promotes the Osteogenesis of BMP-2 in an Absorbable Collagen Sponge.

(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.

Current strategies for integrative cartilage repair.

Osteoarthritis (OA) is a degenerative joint condition characterized by painful cartilage lesions that impair joint mobility. Current treatments such as lavage, microfracture, and osteochondral implantation fail to integrate newly formed tissue with host tissues and establish a stable transition to subchondral bone. Similarly, tissue-engineered grafts that facilitate cartilage and bone regeneration are challenged by how to integrate the graft seamlessly with surrounding host cartilage and/or bone. This review centers on current approaches to promote cartilage graft integration. It begins with an overview of articular cartilage structure and function, as well as degenerative changes to this relationship attributed to aging, disease, and trauma. A discussion of the current progress in integrative cartilage repair follows, focusing on graft or scaffold design strategies targeting cartilage-cartilage and/or cartilage-bone integration. It is emphasized that integrative repair is required to ensure long-term success of the cartilage graft and preserve the integrity of the newly engineered articular cartilage. Studies involving the use of enzymes, choice of cell source, biomaterial selection, growth factor incorporation, and stratified versus gradient scaffolds are therefore highlighted. Moreover, models that accurately evaluate the ability of cartilage grafts to enhance tissue integrity and prevent ectopic calcification are also discussed. A summary and future directions section concludes the review.

Novel organ-slice culturing system to simulate meniscal repair: Proof of concept using a synovium-based pool of meniscoprogenitor cells.

Meniscal injuries can occur secondary to trauma or be instigated by the changes in knee-joint function that are associated with aging, osteo- and rheumatoid arthritis, disturbances in gait, and obesity. Sixty percent of persons over 50 years of age manifest signs of meniscal pathology. The surgical and arthroscopic measures that are currently implemented to treat meniscal deficiencies bring only transient relief from pain and effect but a temporary improvement in joint function. Although tissue-engineering-based approaches to meniscal repair are now being pursued, an appropriate in-vitro model has not been conceived. The aim of this study was to develop an organ-slice culturing system to simulate the repair of human meniscal lesions in vitro. The model consists of a ring of bovine meniscus enclosing a chamber that represents the defect and reproduces its sequestered physiological microenvironment. The defect, which is closed with a porous membrane, is filled with fragments of synovial tissue, as a source of meniscoprogenitor cells, and a fibrin-embedded, calcium-phosphate-entrapped depot of the meniscogenic agents BMP-2 and TGF-ß1. After culturing for 2 to 6 weeks, the constructs were evaluated histochemically and histomorphometrically, as well as immunohistochemically, for the apoptotic marker caspase 3 and collagen types I and II. Under the defined conditions, the fragments of synovium underwent differentiation into meniscal tissue, which bonded with the parent meniscal wall. Both the parent and the neoformed meniscal tissue survived the duration of the culturing period without significant cell losses. The concept on which the in-vitro system is based was thus validated. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1588-1596, 2016.

Activating enhancer binding protein 2 epsilon (AP-2ε)-deficient mice exhibit increased matrix metalloproteinase 13 expression and progressive osteoarthritis development.

INTRODUCTION: The transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e(-/-)) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e(-/-) mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice. METHODS: Murine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e(-/-) mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels. RESULTS: Only minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e(-/-) mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e(-/-) cartilage explants. OA progression was significantly enhanced in the Tfap2e(-/-) mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e(-/-) articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT. CONCLUSIONS: We reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.

Age-Independent Cartilage Generation for Synovium-Based Autologous Chondrocyte Implantation.

The articular cartilage layer of synovial joints is commonly lesioned by trauma or by a degenerative joint disease. Attempts to repair the damage frequently involve the performance of autologous chondrocyte implantation (ACI). Healthy cartilage must be first removed from the joint, and then, on a separate occasion, following the isolation of the chondrocytes and their expansion in vitro, implanted within the lesion. The disadvantages of this therapeutic approach include the destruction of healthy cartilage-which may predispose the joint to osteoarthritic degeneration-the necessarily restricted availability of healthy tissue, the limited proliferative capacity of the donor cells-which declines with age-and the need for two surgical interventions. We postulated that it should be possible to induce synovial stem cells, which are characterized by high, age-independent, proliferative and chondrogenic differentiation capacities, to lay down cartilage within the outer juxtasynovial space after the transcutaneous implantation of a carrier bearing BMP-2 in a slow-release system. The chondrocytes could be isolated on-site and immediately used for ACI. To test this hypothesis, Chinchilla rabbits were used as an experimental model. A collagenous patch bearing BMP-2 in a slow-delivery vehicle was sutured to the inner face of the synovial membrane. The neoformed tissue was excised 5, 8, 11 and 14 days postimplantation for histological and histomorphometric analyses. Neoformed tissue was observed within the outer juxtasynovial space already on the 5th postimplantation day. It contained connective and adipose tissues, and a central nugget of growing cartilage. Between days 5 and 14, the absolute volume of cartilage increased, attaining a value of 12 mm(3) at the latter juncture. Bone was deposited in measurable quantities from the 11th day onwards, but owing to resorption, the net volume did not exceed 1.5 mm(3) (14th day). The findings confirm our hypothesis. The quantity of neoformed cartilage that is deposited after only 1 week within the outer juxtasynovial space would yield sufficient cells for ACI. Since the BMP-2-bearing patches would be implanted transcutaneously in humans, only one surgical or arthroscopic intervention would be called for. Moreover, most importantly, sufficient numbers of cells could be generated in patients of all ages.

How best to preserve and reveal the structural intricacies of cartilaginous tissue.

No single processing technique is capable of optimally preserving each and all of the structural entities of cartilaginous tissue. Hence, the choice of methodology must necessarily be governed by the nature of the component that is targeted for analysis, for example, fibrillar collagens or proteoglycans within the extracellular matrix, or the chondrocytes themselves. This article affords an insight into the pitfalls that are to be encountered when implementing the available techniques and how best to circumvent them. Adult articular cartilage is taken as a representative pars pro toto of the different bodily types. In mammals, this layer of tissue is a component of the synovial joints, wherein it fulfills crucial and diverse biomechanical functions. The biomechanical functions of articular cartilage have their structural and molecular correlates. During the natural course of postnatal development and after the onset of pathological disease processes, such as osteoarthritis, the tissue undergoes structural changes which are intimately reflected in biomechanical modulations. The fine structural intricacies that subserve the changes in tissue function can be accurately assessed only if they are faithfully preserved at the molecular level. For this reason, a careful consideration of the tissue-processing technique is indispensable. Since, as aforementioned, no single methodological tool is capable of optimally preserving all constituents, the approach must be pre-selected with a targeted structure in view. Guidance in this choice is offered.

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress.

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.