In osteoarthritis, chondrocytes acquire a
Purpose:. This study attempts to establish whether biomechanical manipulation through distraction can result in fracture union. Method:. A retrospective clinical audit of 15 patients with delayed or
Introduction: Metastases in multiple myeloma are typically lytic and when non-union occurs it is usually atrophic. Methods: We report a lady of 67 years who was diagnosed with myeloma 9 years previously. She presented with a sudden onset of pain in her right forearm. Plain radiographs demonstrated a lytic lesion typical of multiple myeloma with an undisplaced pathological fracture in her right ulna. The fracture was treated in a short arm cast for 6 weeks and then by mobilisation. The underlying bone deposit was treated subsequently by external beam irradiation. Results: Nine months later she was re-referred to the orthopaedic oncology service with marked forearm pain particularly on rotation. Radiographs demonstrated a
During fracture repair, a number of growth factors and cytokines are present at elevated levels at the fracture site such as Transforming Growth Factor Beta (TGF-), Fibroblast Growth Factor (FGF) and Platelet Derived Growth Factor (PDGF). The aim of the study was to investigate the presence of these growth factors in healing fractures and fracture non-unions, in order to test the hypothesis that atrophic non-unions express a lower level of growth factors than
Purpose. To assess the efficacy of the treatment of the aseptic
Introduction and Aims: Distraction osteogenesis can be used to stimulate healing in
This study aimed to investigate the effect of ATDC5 chondrocytes were cultured in insulin-transferrin-selenium medium to induce differentiation. Cells were transfected with pcDNA3.0 plasmids with either a wild-type (WT) or mutated (MUT) Aims
Methods
Tibial nonunion represents a spectrum of conditions
which are challenging to treat, and optimal management remains unclear
despite its high rate of incidence. We present 44 consecutive patients
with 46 stiff tibial nonunions, treated with hexapod external fixators
and distraction to achieve union and gradual deformity correction.
There were 31 men and 13 women with a mean age of 35 years (18 to
68) and a mean follow-up of 12 months (6 to 40). No tibial osteotomies
or bone graft procedures were performed. Bony union was achieved
after the initial surgery in 41 (89.1%) tibias. Four persistent
nonunions united after repeat treatment with closed hexapod distraction,
resulting in bony union in 45 (97.8%) patients. The mean time to
union was 23 weeks (11 to 49). Leg-length was restored to within
1 cm of the contralateral side in all tibias. Mechanical alignment
was restored to within 5° of normal in 42 (91.3%) tibias. Closed
distraction of stiff tibial nonunions can predictably lead to union
without further surgery or bone graft. In addition to generating
the required distraction to achieve union, hexapod circular external
fixators can accurately correct concurrent deformities and limb-length
discrepancies. Cite this article:
This review is aimed at clinicians appraising
preclinical trauma studies and researchers investigating compromised bone
healing or novel treatments for fractures. It categorises the clinical
scenarios of poor healing of fractures and attempts to match them
with the appropriate animal models in the literature. We performed an extensive literature search of animal models
of long bone fracture repair/nonunion and grouped the resulting
studies according to the clinical scenario they were attempting
to reflect; we then scrutinised them for their reliability and accuracy
in reproducing that clinical scenario. Models for normal fracture repair (primary and secondary), delayed
union, nonunion (atrophic and hypertrophic), segmental defects and
fractures at risk of impaired healing were identified. Their accuracy
in reflecting the clinical scenario ranged greatly and the reliability
of reproducing the scenario ranged from 100% to 40%. It is vital to know the limitations and success of each model
when considering its application.
Osteoarthritis (OA) is the most common joint disease, which is characterized by a progressive loss of proteoglycans and the destruction of extracellular matrix (ECM), leading to a loss of cartilage integrity and joint function. During OA development, chondrocytes alter ECM synthesis and change their gene expression profile including upregulation of
Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies. Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used. Data showed a strong upregulation of
During OA the homeostasis of healthy articular chondrocytes is dysregulated, which leads to a phenotypical transition of the cells, further influenced by external stimuli. Chondrocytes sense those stimuli, integrate them at the intracellular level and respond by modifying their secretory and molecular state. This process is controlled by a complex interplay of intracellular factors. Each factor is influenced by a myriad of feedback mechanisms, making the prediction of what will happen in case of external perturbation challenging. Hampering the
Introduction. Cell-based therapy is needed to overcome the lacking intrinsic ability of cartilage to heal. Generating cartilage tissue from human bone marrow-derived stromal cells (MSC) is limited by up-regulation of COL10, ALP and other hypertrophy markers in vitro and calcifying cartilage at heterotopic sites in vivo. MSC
Aims. The aim of our study is to investigate the effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage (MCC) of growing rabbits. Methods. A total of 64 ten-day-old rabbits were randomly divided into two groups according to dietary hardness: normal diet group (pellet) and soft diet group (powder). In each group, the rabbits were further divided into four subgroups by feeding time: two weeks, four weeks, six weeks, and eight weeks. Animals would be injected 5-bromo-2′-deoxyuridine (BrdU) every day for one week before sacrificing. Histomorphometric analysis of MCC thickness was performed through haematoxylin and eosin (HE) staining. Immunochemical analysis was done to test BrdU and Notch-1. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure expression of Notch-1, Jagged-1, and Delta-like 1 (Dll-1). Results. The thickness of MCC in the soft diet group was thinner than the one in normal diet group. Notch-1 was restricted in fibrous layer, proliferative layer, and
Abstract. Introduction. Recurrent groin pain following periacetabular osteotomy (PAO) is a challenging problem. The purpose of our study was to evaluate the position and dynamics of the psoas tendon as a potential cause for recurrent groin pain following PAO. Methods. Patients with recurrent groin pain following PAO were identified from a single surgeon series. A total of 13 patients with 18 hips (4.7%) out of a 386 PAO, had recurrent groin pain. Muscle path of the psoas tendon was accurately represented using 3D models from CT data were created with Mimics software. A validated discrete element model using rigid body springs was used to predict psoas tendon movement during hip circumduction and walking. Results. Five out of the 18 hips did not show any malformations at the osteotomy site. Thirteen hips (72%) showed malformation secondary to callus at the superior pubic ramus. These were classified into: osteophytes at the osteotomy site,
Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms. Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2. -/-. ). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR. Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over
Virtual mechanical testing is a method for measuring bone healing using finite element models built from computed tomography (CT) scans. Previously, we validated a dual-zone material model for ovine fracture callus that differentiates between mineralized woven bone and soft tissue based on radiodensity. 1. The objective of this study was to translate the dual-zone material model from sheep to two important clinical scenarios: human tibial fractures in early-stage healing and late-stage nonunions. CT scans for N = 19 tibial shaft fractures were obtained prospectively at 12 weeks post-op. A second group of N = 33 tibial nonunions with CT scans were retrospectively identified. The modeling techniques were based on our published method. 2. The dual-zone material model was implemented for humans by performing a cutoff sweep for both the 12-week and nonunion groups. Virtual torsional rigidity (VTR) was calculated as VTR = ML/φ [N-m. 2. /°], where M is the moment reaction, L is the diaphyseal segment length, and φ is the angle of twist. As the soft tissue cutoff was increased, the rigidity of the clinical fractures decreased and soft tissue located within the fracture gaps produced higher strains that are not predicted without the dual zone approach. The structural integrity of the nonunions varied, ranging from very low rigidities in atrophic cases to very high rigidities in highly calcified
The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4). We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes. We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells. We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to
The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a
INTRODUCTION. The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted
Introduction. Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA. Methods. Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and
Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, we report the manufacturing of engineered and devitalized human
Nonunions occur in situations with interrupted fracture healing process and indicate conditions where the fracture has no potential to heal without further intervention. Per definition, no healing is detected nine months post operation and there is no visible progress of healing over the last three months. The classification of nonunions as
INTRODUCTION. The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted ossification of the generated tissue due to endochondral ossification. Our in vitro data show that celecoxib is able to suppress the
Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a
INTRODUCTION. Endochondral ossification in the growth plate is directly responsible for skeletal growth and its de novo bone-generating activity. Growth plates are vulnerable to disturbances that may lead to abnormal skeletal development. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics but have been reported to impair endochondral ossification-driven fracture healing. Despite the general awareness that NSAIDs affect endochondral ossification, the consequences of NSAIDs on skeletal development are unknown. We hypothesise that the NSAID celecoxib leads to impaired growth plate development and consequently impairs skeletal development. METHODS. Healthy skeletally immature (5 weeks old) C57BL/6 mice were treated for ten weeks with celecoxib (daily oral administration 10 mg/kg) or placebo (water) (institutional approval 2013–094) (n=12 per group). At 15 weeks postnatally, total growth plate thickness, the thickness of specific growth plate zones, (immuno)histological analysis of extracellular matrix composition in the growth plate, cell number and cell size, longitudinal bone growth and bone micro-architecture by micro-CT were analysed. Inhibition of COX-2 activity was confirmed by determining PGE2 levels in plasma using an ELISA. RESULTS. No significant difference in total growth plate thickness or thickness of the resting zone, proliferative or
Introduction. A significant burden of disease exists with respect to critical sized bone defects; outcomes are unpredictable and often poor. There is no absolute agreement on what constitutes a “critically-sized” bone defect however it is widely considered as one that would not heal spontaneously despite surgical stabilisation, thus requiring re-operation. The aetiology of such defects is varied. High-energy trauma with soft tissue loss and periosteal stripping, bone infection and tumour resection all require extensive debridement and the critical-sized defects generated require careful consideration and strategic management. Current management practice of these defects lacks consensus. Existing literature tells us that tibial defects 25mm or great have a poor natural history; however, there is no universally agreed management strategy and there remains a significant evidence gap. Drawing its origins from musculoskeletal oncology, the Capanna technique describes a hybrid mode of reconstruction. Mass allograft is combined with a vascularised fibula autograft, allowing the patient to benefit from the favourable characteristics of two popular reconstruction techniques. Allograft confers initial mechanical stability with autograft contributing osteogenic, inductive and conductive capacity to encourage union. Secondarily its inherent vascularity affords the construct the ability to withstand deleterious effects of stressors such as infection that may threaten union. The strengths of this hybrid construct we believe can be used within the context of critical-sized bone defects within tibial trauma to the same success as seen within tumour reconstruction. Methodology. Utilising the Capanna technique in trauma requires modification to the original procedure. In tumour surgery pre-operative cross-sectional imaging is a pre-requisite. This allows surgeons to assess margins, plan resections and order allograft to match the defect. In trauma this is not possible. We therefore propose a two-stage approach to address critical-sized tibial defects in open fractures. After initial debridement, external fixation and soft tissue management via a combined orthoplastics approach, CT imaging is performed to assess the defect geometry, with a polymethylmethacrylate (PMMA) spacer placed at index procedure to maintain soft tissue tension, alignment and deliver local antibiotics. Once comfortable that no further debridement is required and the risk of infection is appropriate then 3D printing technology can be used to mill custom jigs. Appropriate tibial allograft is ordered based on CT measurements. A pedicled fibula graft is raised through a lateral approach. The peroneal vessels are mobilised to the tibioperoneal trunk and passed medially into the bone void. The cadaveric bone is prepared using the custom jig on the back table and posterolateral troughs made to allow insertion of the fibula, permitting some
Introduction and Objective. Current cartilage repair strategies lack adequate tissue integration capacity and often present mechanical failure at the graft-to-host tissue junction. The design of multilayered osteochondral tissue engineering (TE) constructs is an attractive approach to overcome these problems. However, calcium ion-release from resorbable bone-replacement materials was suggested to compromise chondrogenic differentiation of adjacent cartilage tissue and it is unclear whether articular chondrocytes (AC) or mesenchymal stroma cells (MSC) are more sensitive to such conditions. Aim of the study was to compare how elevated calcium levels affect cartilage matrix production during re-differentiation of AC versus chondrogenic differentiation of MSC. The results of this study will help to identify the ideal cell source for growth of neocartilage adjacent to a calcified bone replacement material for design of multilayered osteochondral TE approaches. Materials and Methods. Expanded human AC and MSC (6–12 donors per group) were seeded in collagen type I/III scaffolds and cultured under standard chondrogenic conditions at control (1.8mM) or elevated (8.0mM) CaCl2 for 35 days. Proteoglycan and collagen production were assessed via radiolabel-incorporation, ELISA, qPCR and Western blotting. Differences between groups or cell types were calculated using the non-parametric Wilcoxon or Mann-Whitney U test, respectively, with p < 0.05 considered significant. Results. Elevated calcium significantly reduced GAG synthesis (63% of control, p=0.04) and chondrogenic marker expression of AC, lowering the GAG/DNA content (47% of control, p=0.004) and collagen type II deposition (24% of control, p=0.05) of neocartilage compared to control conditions. Opposite, at elevated calcium levels MSC-derived chondrocytes significantly increased GAG synthesis (130% of control, p=0.02) and collagen type II content (160% of control, p=0.03) of cartilage compared to control tissue. Chondrogenic and
Heterotopic ossi?cation is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossi?cations are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossi?cation on a molecular level, therefore we aimed to determine whether indomethacin might influence heterotopic ossi?cation via impairing the chondrogenic phase of endochondral ossification. ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE2 levels in all experiments, and was determined by specific PGE2 ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome. Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a dose-dependent decrease in gene- and protein expression of chondrogenic and
Chondromodulin-I (ChM-I) is a bifunctional autocrine regulator of cartilage, initially isolated from fetal bovine epiphyseal cartilage. 1. ChM-I stimulates matrix synthesis of chondrocytes, but inhibits the growth of endothelial cells. 1,. 2. thus ChM-I may be one of the anti-angiogenic molecules present in cartilage. In fetal bovine bone, ChM-I was expressed by all epiphyseal and growth plate chondrocytes except
Dysplasia Epiphysealis Hemimelica (DEH) also known as Trevor's Disease is a rare developmental disorder resulting in cartilaginous overgrowth of the epiphysis of long bones. DEH is usually diagnosed in children between two and eight years old and it is three times more often diagnosed in boys. The most reported complaints are pain, limitation in range of motion, and deformity or swelling of the affected joint. Treatment of symptomatic lesions consists of surgical resection of the lesion, resulting in good long-term results. Based on histological evaluation, DEH is often described as an osteochondroma or an osteochondroma-like lesion, although there are clinical, radiological and genetic differences between DEH and osteochondromas. To investigate the hypothesis that DEH and osteochondromas are histologically identical, two cases of DEH and two cases of osteochondromas in patients with Hereditary Multiple Osteochondroma (HMO) are compared at histological level. Tissue samples from patients with a histopathologically confirmed diagnosis of DEH were compared with two age and gender matched patients diagnosed with HMO. After tissue sampling and processing, (immuno)histological stainings were performed for Collagen type II, Collagen type X, Sox-9 and Safranin-O. Histologically, clumping of chondrocytes in a fibrillar matrix, a thick disorganized cartilage cap and ossification centres with small amounts of unresorbed cartilage were observed in DEH. In contrast, chondrocyte organisation in cartilage of osteochondromas displays characteristics of the normal growth plate. In addition, differences in expression of collagen type II, collagen type X and Sox9 were observed. Collagen type II was expressed in the extracellular matrix surrounding proliferative and
Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper
Introduction and Objective. Klinefelter Syndrome (KS, karyotype 47,XXY) is the most frequent chromosomal aneuploidy in males, as well as the most common cause of infertility in men. Patients suffer from a lack of testosterone, i.e. hypergonadotropic hypogonadism provoking infertility, but KS men also show an increased predisposition to osteoporosis and a higher risk of bone fracture. In a mouse model for human KS, bone analysis of adult mice revealed a decrease in bone mass that could not be rescued by testosterone replacement, suggesting a gene dosage effect originating from the supernumerary X-chromosome on bone metabolism. Usually, X chromosome inactivation (XCI) compensates for the dosage imbalance of X-chromosomal genes between sexes. Some studies suggested that expression of genes that escape silencing of the supernumerary X-chromosome (e.g. androgen receptor) has an impact on sex differences, but may also cause pathological changes in males. As a promising new such candidate for a musculoskeletal escape gene, we identified the integral membrane protein (ITM) 2a, which is encoded on the X-chromosome and related to enchondral ossification. The aim of the project was to characterize systemic bone loss in the course of aging in our KS mouse model, and whether the supernumerary X-chromosome causes differences in expression of genes related to bone development. Materials and Methods. Bone structure of 24 month (=aged) old male wild type (WT) and 41, XXY mice (B6Ei.Lt-Y) were analysed by μCT. Afterwards bones were paraffin embedded and cut. In addition, tissue of brain, liver, kidney, lung and heart were also isolated and embedded for IHC staining. Using an anti-ITM2a antibody, expression and cellular localization of ITM2a was evaluated. IHC was also performed on musculoskeletal tissue of WT embryos (E18.5) and neonatal mice to determine possible age-related differences. Results. In 24 month old mice, the analysis of the lumbar vertebrae revealed a significantly lower BV/TV, trabecular bone volume and trabecular number in the XXY- group compared to WT. Trabecular thickness appeared lower but did not reach significance, with the cortical thickness being significantly higher in the XXY- group. High expression of ITM2a was detected in bone slices of both karyotypes in the chondrocytes inside the growth plate, as well as in megakaryocytes and leucocytes as well as endothelial cells of blood vessels inside the bone marrow. Osteocytes, along with erythrocytes and erythropoetic stem cells were negative for ITM2a. Other organs that showed ITM2a positive staining were kidney (blood vessels), heart (muscle) and brain (different structures). Liver and lung tissue were negative for ITM2a. No obvious difference in the intensity of the ITM2a-expression was observed between the WT and the XXY-karyotype. Analyses of embryotic bone tissue (WT) showed high expression of ITM2a in proliferating,
Introduction. Current cell-based treatments and marrow stimulating techniques to repair articular cartilage defects are limited in restoring the tissue in its native composition. Despite progress in cartilage tissue engineering and chondrogenesis in vitro, the main limitation of this approach is the progression towards hypertrophy during prolonged culture in pellets or embedded in biomaterials. The objectives of this study were (A) to compare human bone marrow-derived mesenchymal stromal cells (hMSC) chondrogenesis and hypertrophy in pellet culture from single cells or cell spheroids and (B) to investigate the effect of tyramine-modified hyaluronic acid (THA) and collagen I (Col) content in composite hydrogels on the chondrogenesis and hypertrophy of encapsulated hMSC spheroids. Materials and Methods. Pellet cultures were prepared either from hMSC single cells (250’000 cells/pellet) or hMSC spheroids (282 cells/spheroid) at the same final cell concentration (250’000 cells/pellet = 887 spheroids/pellet). The effect of polymer concentration on encapsulated hMSC spheroids (887 spheroids/hydrogel) was investigated in THA-Col hydrogels (50μl) at the following concentrations (THA-Col mg/ml): Group (1) 12.5–2.5, (2) 16.7–1.7, (3) 12.5–1.7, (4) 16.7–2.5 mg/ml. All samples were cultured for 21 days in standard chondrogenic differentiation medium containing 10ng/ml TGF-β1. Chondrogenic differentiation and hypertrophy of both pellet cultures and hMSCs spheroids encapsulated in THA-Col were analysed using gene expression analysis (Aggrecan (ACAN), COL1A1, COL2A1, COL10A1), dimethylmethylene-Blue assay to quantify glycosaminoglycans (GAGs) retained in the samples and (immuno-) histological staining (Safranin-O, collagen II, aggrecan) on day 1 and day 21 (n=3 donors). Results. The culture of hMSCs in pellets based on single cells or spheroids resulted in an increase in chondrogenic-associated markers COL2A1 (2’900–3’400-fold) and ACAN (45–47-fold) compared to respective samples on day 1 in both groups. GAGs increased in spheroid pellets to 21.2±3.4 mg/ml and in single cell pellets to 20.8±6.6 mg/ml on day 21. Comparing the levels of
Introduction: In the growth plate, chondrocyte swelling (hypertrophy) is a crucial event during endochondral ossification and bone lengthening, accounting for ~80% of the increase in bone length (. 1. ,. 3. ). The swelling is dramatic (~10x) and closely regulated. Failure of chondrocyte hypertrophy may underlie the chondrodysplasias of the vertebrate skeleton (. 1. ). However, the mechanisms which control cell swelling are poorly understood although there must be a key role for chondrocyte osmolyte transporters which are sensitive to an increase in cell volume. We have used confocal scanning laser microscopy (CLSM) to study volume regulation by living in situ growth plate chondrocytes at varying degrees of hypertrophy. Methods: Bovine growth plates were taken from the ends of young (~12d) bovine ribs. In situ growth plate chondrocytes at the proliferative through to
Introduction. The hematoma occurring at a fracture site is known to play an important role in fracture healing. Previously, we demonstrated that fracture hematoma contained multilineage mesenchymal progenitor cells. On the other hand, the process of fracture healing is associated by two different mechanisms, intramembranous and endochondral. However, there are no reports proving the details about cellular analysis in the process of endochondoral ossification. Hypothesis. We hypothesized that one of the cell origins for endochondral ossification after fracture was hematoma. Materials & Methods. Fracture hematoma was obtained during osteosynthesis. Hematoma-derived cells were isolated and cultured for 5-weeks of chondrogenic induction followed by 2-weeks
Osteoarthritis (OA) is a progressive and degenerative joint disease resulting in changes to articular cartilage. In focal early OA defects, autologous chondrocyte implantation (ACI) has a 2-fold failure rate due to poor graft integration and presence of inflammatory factors (e.g. Interleukin-1β). Bone marrow derived mesenchymal stem cells (MSCs) are an alternative cell source for cell-based treatments due to their chondrogenic capacity, though in vivo implantation leads to bone formation. In vivo, chondrocytes reside under an oxygen tension between 2–7% oxygen or physioxia. Physioxia enhances MSC chondrogenesis with reduced
The December 2015 Trauma Roundup. 360 . looks at: Delay to surgery in hip fracture; Hexapod fixators in the management of
Summary. Indomethacin has differential effects on chondrogencic outcome depending on differentiation stage. Introduction. Heterotopic ossification (HO) is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. The standard treatment to prevent HO is administration of the NSAID indomethacin. HOs are described to develop via endochondral ossification. As it is currently unknown how indomethacin prevents HO, we aimed to define whether indomethacin might influence HO via impairing the chondrogenic phase of endochondral ossification. Materials. ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE. 2. levels in all experiments, and was determined by specific PGE. 2. ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome. Results. A dose-dependent decrease in expression of Col2a1, Col10a1 and GAG content was observed when progenitor ATDC5 cells differentiating in the chondrogenic lineage were treated with increasing concentrations of indomethacin. These results were confirmed on primary hBMSCs and ex vivo periosteal agarose cultures. Even when
Purpose of the study: Data concerning the teres minor in the context of rotator cuff tears is scarce despite the fact that this muscle plays a crucial role in the event of an infraspinaus tear. The purpose of this study was to analyse the computed tomography and magnetic resonance images of the teres minor muscle in rotator cuff tears. Material and method: The aspect of the teres minor muscle was studied in 1624 shoulders with rotator cuff tears. The axial and sagittal CT or MRI aspect was noted as normal,
Summary Statement. This study reports that hMSC can be manipulated in order to engineer a bone organ, characterised by mature osseous and vascular components and capable to recruit, host and maintain functional HSCs. Introduction. Bone tissue engineering strategies are typically based on methods involving adult human Mesenchymal Stromal Cells (hMSC) in a process resembling intramembranous ossification. However, most bones develop and repair through endochondral ossification. In addition, endochondral ossification presents several advantages for regenerative purposes such as osteogenic activity, capability to drive formation of the Hematopoietic Stem Cell (HSC) niche, resistance to hypoxia, intrinsic vasculogenic potential and, consequently, efficiency of engraftment. In this study, we aimed at developing an endochondral bone organ model characterised by functional osseous and hematopoietic compartments by using hMSC. Materials & Methods. Expanded hMSC were seeded onto 8 mm diameter, 2 mm thick collagen sponges (UltrafoamTM, Davol Inc.), cultured for vitro under defined chondrogenic (3 weeks) and
The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, and is important for joint long-term function. Previous studies have shown that TGF-β/Alk5 signaling upregulating PRG4 expression maintains articular cartilage homeostasis. However, the exact role and molecular mechanism of TGF-β signaling in SFZ of articular cartilage homeostasis are still lacking. In this study, a combination of in vitro and in vivo approaches were used to elucidate the role of Alk5 signaling in maintaining the SFZ of articular cartilage and preventing osteoarthritis initiation. Mice with inducible cartilage SFZ-specific deletion of Alk5 were generated to assess the role of Alk5 in OA development. Alterations in cartilage structure were evaluated histologically. The chondrocyte apoptosis and cell cycle were detected by TUNEL and Edu staining, respectively. Isolation, culture and treatment of SFZ cells, the expressions of genes associated with articular cartilage homeostasis and TGF-β signaling were analyzed by qRT-PCR. The effects of TGF-β/Alk5 signaling on proliferation and differentiation of SFZ cells were explored by cells count and alcian blue staining. In addition, SFZ cells isolated from C57 mice were cultured in presence of TGF-β1 or SB505124 for 7 days and transplanted subcutaneously in athymic mice. Postnatal cartilage SFZ-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. qRT-PCR and IHC results confirmed that Alk5 gene was effectively deleted in articular cartilage SFZ cells. Next, the PRG4-positive cells in articular cartilage SFZ were significantly decreased in Alk5 cKO mice compared with those in Cre-negative control mice. The mRNA expression of Aggrecan and Col2 were decreased, meanwhile, expression of Mmp13 and Adamts5 were significantly increased in articular cartilage SFZ cells of Alk5 cKO mice. In addition, Edu and TUNEL staining results revealed that slow-cell cycle cell number and increase the apoptosis positive cell in articular cartilage SFZ of Alk5 cKO mice compared with Cre-negative mice, respectively. Furthermore, all groups of SFZ cells formed ectopic solid tissue masses 1 week after transplantation. Histological examination revealed that the TGF-β1-pretreated tissues was composed of small and round cells and was positive for alcian blue staining, while the SB505124-pretreated tissue contained more
Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model. Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/
Purpose of the study: The process of bone lengthening involves three phases: a latence period, distraction, then healing. The healing phase required stability maintained by an external fixator (EF) for 1.16 months/cm lengthening. This time exposes the patient to serious complications. The objective is to accelerate the healing phase in order to shorten the time the patient has to wear the EF. The effect of BMP on osteogenesis in distraction remains a controversial issue. This work was conducted to evaluate the benefit provided by rhBMP-2 for healing the regenerate bone after distraction. Material and methods: Thirty-nine subadult male rabbits were selected at random. On day 0, a tibial osteotomy was performed followed by installation of a M101 EF. After the latency period of seven days, the distraction began at the rate of 0.5mm/12 h for 21 days. At day 28, at the end of distraction, a new operation was performed and three groups of 13 individuals were created at random. The first group received no material, the second a collagen type 1 sponge, and the third group a collagen type 1 sponge soaked in 100 μg/kg rhBMP-2. The animals were monitored with x-rays, absorptiometry and ultrasound for the qualitative and quantitative analysis. Histological and biomechanical analyses were performed at two months. Results: Our complication rate was 41%. Qualitative analysis of the x-rays showed, in group 3, the development of more or less voluminous and dense, sometimes
Aim: There have been increasingly publications about the complicated disease of patello-femoral joints after total knee arthroplasty (TKA). We have treated soft tissue impingement under the patella after TKA by arthroscopic surgery and investigated the findings and efficacy of the treatment. Materials and Methods: 6 patients and 8 knees which showed soft tissue impingement of patello-femoral joints after TKA. Surgical arthroscopy was performed and impinging soft tissues were classified and the efficacy of arthroscopic treatment were evaluated. Results: We classified the patients with soft tissue impingement under the patella into three groups: (I) patellar clunk syndrome; the isolated fibrous nodule located suprapatellar lesion, without the other fibrous tissues causing the impingement, (II) impinging
Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future.
While mesenchymal stromal cells (MSCs) are a very attractive cell source for cartilage regeneration, an inherent tendency to undergo
A 30-year-old man presented with pain and limitation of movement of the right hip. The symptoms had failed to respond to conservative treatment. Radiographs and CT scans revealed evidence of impingement between the femoral head-neck junction and an abnormally large anterior inferior iliac spine. Resection of the
Introduction. The results of repair and reconstruction of lesions found in the abductor muscles and tendons during posterior approach to primary total hip arthroplasty (THA) were reported in 2018. During the course of this series it became apparent that the extent of damage in the abductor tendons and muscles usually was obscured by the
The changes occurring in ligamentum flavum in lumbar spine stenosis are a matter of long–standing controversy. More recently, some studies showed that the posterior spinal structures, including hypertrophied ligamentum flavum, play a major role in the pathogenesis of the lumbar stenosis. To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Samples of ligamentum flavum were collected from 4 patients undergoing surgery for lumbar stenosis (mean age 47.2 years). Cell cultures were obtained from each patient and maintained in Dulbecco’s modified essential medium-10% fetal calf serum. Cell characterization was histochemically (Gomori’s and von Kossa staining), immunohistochemically (anti-type I, -type II, -type III and -type X collagen, anti-S100 protein, anti-fibronectin, anti-osteonectin and anti-osteocalcin), biochemically (cAMP activity after human parathyroid hormone stimulation) assessed. Samples collected from 2 age-matched patients who underwent surgery for lumbar fractures were used as controls. Stenotic ligamentum flavum cells expressed high levels of alkaline phosphatase activity and produced a mineralized matrix rich in type I, type III and type X collagen, fibronectin, osteonectin, and osteocalcin. Stimulation with parathyroid hormone increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. Staining for type II and type X collagen, and S-100 protein reflected the proliferation of