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.

Microbeam Radiation Therapy Controls Local Growth of Radioresistant Melanoma and Treats Out-of-Field Locoregional Metastasis.

PURPOSE: Synchrotron-generated microbeam radiation therapy (MRT) represents an innovative preclinical type of cancer radiation therapy with an excellent therapeutic ratio. Beyond local control, metastatic spread is another important endpoint to assess the effectiveness of radiation therapy treatment. Currently, no data exist on an association between MRT and metastasis. Here, we evaluated the ability of MRT to delay B16F10 murine melanoma progression and locoregional metastatic spread. METHODS AND MATERIALS: We assessed the primary tumor response and the extent of metastasis in sentinel lymph nodes in 2 cohorts of C57BL/6J mice, one receiving a single MRT and another receiving 2 MRT treatments delivered with a 10-day interval. We compared these 2 cohorts with synchrotron broad beam-irradiated and nonirradiated mice. In addition, using multiplex quantitative platforms, we measured plasma concentrations of 34 pro- and anti-inflammatory cytokines and frequencies of immune cell subsets infiltrating primary tumors that received either 1 or 2 MRT treatments. RESULTS: Two MRT treatments were significantly more effective for local control than a single MRT. Remarkably, the second MRT also triggered a pronounced regression of out-of-radiation field locoregional metastasis. Augmentation of CXCL5, CXCL12, and CCL22 levels after the second MRT indicated that inhibition of melanoma progression could be associated with increased activity of antitumor neutrophils and T-cells. Indeed, we demonstrated elevated infiltration of neutrophils and activated T-cells in the tumors after the second MRT. CONCLUSIONS: Our study highlights the importance of monitoring metastasis after MRT and provides the first MRT fractionation schedule that promotes local and locoregional control with the potential to manage distant metastasis.

Targeted Accumulation of Macrophages Induced by Microbeam Irradiation in a Tissue-Dependent Manner.

Radiation therapy (RT) is a vital component of multimodal cancer treatment, and its immunomodulatory effects are a major focus of current therapeutic strategies. Macrophages are some of the first cells recruited to sites of radiation-induced injury where they can aid in tissue repair, propagate radiation-induced fibrogenesis and influence tumour dynamics. Microbeam radiation therapy (MRT) is a unique, spatially fractionated radiation modality that has demonstrated exceptional tumour control and reduction in normal tissue toxicity, including fibrosis. We conducted a morphological analysis of MRT-irradiated normal liver, lung and skin tissues as well as lung and melanoma tumours. MRT induced distinct patterns of DNA damage, reflecting the geometry of the microbeam array. Macrophages infiltrated these regions of peak dose deposition at variable timepoints post-irradiation depending on the tissue type. In normal liver and lung tissue, macrophages clearly demarcated the beam path by 48 h and 7 days post-irradiation, respectively. This was not reflected, however, in normal skin tissue, despite clear DNA damage marking the beam path. Persistent DNA damage was observed in MRT-irradiated lung carcinoma, with an accompanying geometry-specific influx of mixed M1/M2-like macrophage populations. These data indicate the unique potential of MRT as a tool to induce a remarkable accumulation of macrophages in an organ/tissue-specific manner. Further characterization of these macrophage populations is warranted to identify their organ-specific roles in normal tissue sparing and anti-tumour responses.

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.

Synchrotron Microbeam Radiation Therapy for the Treatment of Lung Carcinoma: A Preclinical Study.

PURPOSE: In the past 3 decades, synchrotron microbeam radiation therapy (S-MRT) has been shown to achieve both good tumor control and normal tissue sparing in a range of preclinical animal models. However, the use of S-MRT for the treatment of lung tumors has not yet been investigated. This study is the first to evaluate the therapeutic efficacy of S-MRT for the treatment of lung carcinoma, using a new syngeneic and orthotopic mouse model. METHODS AND MATERIALS: Lewis Lung carcinoma-bearing mice were irradiated with 2 cross-fired arrays of S-MRT or synchrotron broad-beam (S-BB) radiation therapy. S-MRT consisted of 17 microbeams with a width of 50 µm and center-to-center spacing of 400 µm. Each microbeam delivered a peak entrance dose of 400 Gy whereas S-BB delivered a homogeneous entrance dose of 5.16 Gy (corresponding to the S-MRT valley dose). RESULTS: Both treatments prolonged the survival of mice relative to the untreated controls. However, mice in the S-MRT group developed severe pulmonary edema around the irradiated carcinomas and did not have improved survival relative to the S-BB group. Subsequent postmortem examination of tumor size revealed that the mice in the S-MRT group had notably smaller tumor volume compared with the S-BB group, despite the presence of edema. Mice that were sham-implanted did not display any decline in health after S-MRT, experiencing only mild and transient edema between 4 days and 3 months postirradiation which disappeared after 4 months. Finally, a parallel study investigating the lungs of healthy mice showed the complete absence of radiation-induced pulmonary fibrosis 6 months after S-MRT. CONCLUSIONS: S-MRT is a promising tool for the treatment of lung carcinoma, reducing tumor size compared with mice treated with S-BB and sparing healthy lungs from pulmonary fibrosis. Future experiments should focus on optimizing S-MRT parameters to minimize pulmonary edema and maximize the therapeutic ratio.

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.

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.

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.

TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Chondrogenic differentiation of bovine synovium: bone morphogenetic proteins 2 and 7 and transforming growth factor beta1 induce the formation of different types of cartilaginous tissue.

OBJECTIVE: To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor beta1 (TGFbeta1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. METHODS: Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFbeta1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. RESULTS: BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFbeta1 differed in terms of both cell phenotype and gene expression profiles. CONCLUSION: The 3 tested members of the TGFbeta superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

Expression of cartilage-related genes in bovine synovial tissue.

The synovium contains mesenchymal stem cells with chondrogenic potential. Although synovial and articular cartilage tissue develop from a common pool of mesenchymal cells, little is known about their genetic commonalities. In the present study, the mRNA levels for several cartilage-related proteins, namely, cartilage oligomeric matrix protein (COMP), Sox9, aggrecan, and collagen types I, II, IX, X, and XI, were measured using the real-time polymerase chain reaction. Our data reveal the synovium of calf metacarpal joints to physiologically express not only type I collagen but also COMP, Sox9, aggrecan, and collagen types X and XI. The mRNA levels for the latter five proteins lie between 2% and 15% of those in articular cartilage. We speculate that these genes are being expressed by chondroprogenitor cells, whose presence in the synovium reflects a common ontogenetic phase in the fetal development of this tissue and of articular cartilage.