Karen B. King

Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo.

Reprogramming somatic cells into an ESC‐like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage‐specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS‐derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage‐specific genes. Isolated implants of iPS cell‐derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease. STEM CELLS 2011;29:206–216

The Effects of Collagen Fragments on the Extracellular Matrix Metabolism of Bovine and Human Chondrocytes

Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.

Expression of anchorin CII (cartilage annexin V) in human young, normal adult, and osteoarthritic cartilage.

In its tissue-specific function as a collagen receptor of chondrocytes, cartilage annexin V (anchorin CII) occupies a key position in the organization of the cell-extracellular matrix (ECM) junction for the tissue. The general role of annexin V (Anx V) in other tissues suggests involvement in cellular secretory processes and in regulation of apoptosis. Immunohistochemical analysis of Anx V in growth plate cartilage, confirmed by in situ hybridization, suggests that Anx V is prominently expressed and forms a major constituent of growth plate chondrocytes. Anx V epitopes are also located in the pericellular matrix of hypertrophic cartilage. In adult articular cartilage the expression is downregulated, with the highest levels of immunostaining found in the upper third of the articular cartilage layers and almost no antigen found in the deep layers. Osteoarthritic (OA) cartilage is characterized by a significant upregulation of message and protein throughout the entire depth of the tissue, an accumulation of cytoplasmic annexin V epitopes, and a release of epitopes into the pericellular and interterritorial matrix, in part co-localized with granular structures. Therefore, Anx V expression and tissue distribution may serve as a histological marker for metabolic alterations and for changes in the cellular phenotype associated with OA.

The adverse effects of diabetes on osteoarthritis: update on clinical evidence and molecular mechanisms

Projected increases in the prevalence of both diabetes mellitus (DM) and osteoarthritis (OA) ensure their common co-existence. In an era of increasing attention to personalized medicine, understanding the influence of common comorbidities such as DM should result in improved care of patients with OA. In this narrative review, we summarize the literature addressing the interactions between DM and OA spanning the years from 1962 to 2014. We separated studies depending on whether they investigated clinical populations, animal models, or cells and tissues. The clinical literature addressing the influence of DM on OA and its therapeutic outcomes suggests that DM may augment the development and severity of OA and that DM increases risks associated with joint replacement surgery. The few high quality studies using animal models also support an adverse effect of DM on OA. We review strengths and weaknesses of the common rodent models of DM. The heterogeneous literature derived from studies of articular cells and tissues also supports the existence of biochemical and biomechanical changes in articular tissues in DM, and begins to characterize molecular mechanisms activated in diabetic-like environs which may contribute to OA. Increasing evidence from the clinic and the laboratory supports an adverse effect of DM on the development, severity, and therapeutic outcomes for OA. To understand the mechanisms through which DM contributes to OA, further studies are clearly necessary. Future studies of DM-influenced mechanisms may shed light on general mechanisms of OA pathogenesis and result in more specific and effective therapies for all OA patients.

Change in serum COMP concentration due to ambulatory load is not related to knee OA Status

The aim of this study was to test the hypothesis that a change in serum cartilage oligomeric matrix protein (COMP) concentration is related to joint load during a 30‐min walking exercise in patients with medial compartment knee osteoarthritis (OA) and in age‐matched control subjects. Blood samples were drawn from 42 patients with medial compartment knee OA and from 41 healthy age‐matched control subjects immediately before, immediately after, and 0.5, 1.5, 3.5, and 5.5 h after a 30‐min walking exercise on a level outdoor walking track at self‐selected normal speed. Serum COMP concentrations were determined using a commercial ELISA. Basic time–distance gait variables were recorded using an activity monitor. Joint loads were measured using gait analysis. Serum COMP concentrations increased immediately after the walking exercise (+6.3% and +5.6%; p < 0.001) and decreased over 5.5 h after the exercise (−11.1% and −14.6%; p < 0.040 and p = 0.001) in patients and control subjects, respectively. The magnitude of increase in COMP concentration did not differ between groups (p = 0.902) and did not correlate with any variables describing ambulatory loads at the joints of the lower extremity. These results, taken together with a previous study of a younger healthy population, suggest the possibility that the influence of ambulatory loads on cartilage turnover is dependent on age.

Long-term cyclical in vivo loading increases cartilage proteoglycan content in a spatially specific manner: an infrared microspectroscopic imaging and polarized light microscopy study.

Understanding the changes in collagen and proteoglycan content of cartilage due to physical forces is necessary for progress in treating joint disorders, including those due to overuse. Physical forces in the chondrocyte environment can affect the cellular processes involved in the biosynthesis of extracellular matrix. In turn, the biomechanical properties of cartilage depend on its collagen and proteoglycan content. To understand changes due to physical forces, this study examined the effect of 80 cumulative hours of in vivo cyclical joint loading on the cartilage content of proteoglycan and collagen in the rabbit metacarpophalangeal joint. The forepaw digits of six anesthetized New Zealand White adult female rabbits were repetitively flexed at 1 Hz with an estimated joint contact pressure of 1 to 2 MPa. Joints were collected from loaded and contralateral control specimens, fixed, decalcified, embedded, and thin-sectioned. Sections were examined under polarized light microscopy to identify and measure superficial and mid zone thicknesses of cartilage. Fourier Transform Infrared microspectroscopy was used to measure proteoglycan and collagen contents in the superficial, mid, and deep zones. Loading led to an increase in proteoglycan in the cartilage of all six rabbits. Specifically, there was a 46% increase in the cartilage deep zone (p = 0.003). The collagen content did not change with loading. Joint loading did not change the superficial and mid zone mean thicknesses. We conclude that long-term (80 cumulative hours) cyclical in vivo joint loading stimulates proteoglycan synthesis. Furthermore, stimulation is localized to cartilage regions of high hydrostatic pressure. These data may be useful in developing interventions to prevent overuse injuries or in developing therapies to improve joint function.

The Establishment and Characterization of an Immortal Cell Line With a Stable Chondrocytic Phenotype

A cell line was developed from the transplantable Swarm rat chondrosarcoma (RCS) and has been maintained in continuous monolayer tissue culture for a number of years. This long term‐cultured (LTC) cell line exhibits the morphological and biochemical characteristics of chondrocytes and resembles the RCS tumor by electron and light microscopy. The cell line differs from the original tumor cells in that about 90% of the sulfated macromolecules are retained in the LTC extracellular matrix as compared to 30% by primary cultures of cells from the RCS tumor. An interesting and useful feature of this cell line is that it contains clonal populations of cells which differ in the quality and quantity of matrix produced. Two such clones serve to illustrate the diversity of cell types within the LTC cell line. One termed Rex accumulates an intensely staining matrix around it, while the other, Ng, accumulates a matrix, that remains virtually unstained. The chondrocytic nature and ease of cloning make these cells ideal for biochemical analysis of the chondrocyte and its extracellular matrix. J. Cell. Biochem. 89: 992–1004, 2003.

Veterans With Diabetes Receive Arthroplasty More Frequently and at a Younger Age

BackgroundA future increase in total joint arthroplasties in patients with diabetes seems likely considering the prevalence of osteoarthritis and diabetes mellitus are increasing. However, the rates of arthroplasty in the population of patients with diabetes are unclear.Questions/purposesWe sought to determine whether lower extremity arthroplasties in a veteran population with diabetes is different from a similar population without diabetes. The following specific questions were asked: (1) Is the rate of TKA in veterans with diabetes higher than in those without diabetes? (2) Is the rate of THA in veterans with diabetes higher than in those without diabetes? (3) Are arthroplasty revision rates greater in veterans with diabetes than in veterans without diabetes?MethodsThe US Department of Veterans Affairs Health administrative data from fiscal year 2000 was used to identify persons with primary or secondary TKA or THA. The rate of surgeries among a diabetic population was compared with that among a nondiabetic population.ResultsThe diabetic cohort received total joint arthroplasties at a higher rate than the nondiabetic cohort at all ages younger than 66 years, with a range of odd ratios from 1.3 to 3.4. In answer to our specific questions, (1) the rate of TKA (95% CI, 2.1–3.7), (2) the rate of THA (95% CI, 1.0–2.6), and (3) the rates of arthroplasty revision (95% CI, 0.9–5.8 TKA and 0.7–6.8 THA) were higher in veterans with diabetes. Furthermore, those with diabetes in the youngest age group studied received total joint arthroplasties and revision surgeries at approximately double the rates of those without diabetes.ConclusionsIf these findings hold true for the population as a whole, they imply that clinicians in the United States may see a sharp increase in younger diabetic candidates for joint arthroplasty.Level of EvidenceLevel III, prognostic study. See Guidelines for Authors for a complete description of levels of evidence.

Arthroplasty in veterans: Analysis of cartilage, bone, serum, and synovial fluid reveals differences and similarities in osteoarthritis with and without comorbid diabetes

Osteoarthritis patients with diabetes who receive total knee arthroplasty are more vulnerable to complications, including aseptic loosening and need for revision surgery. To elucidate mechanisms related to arthroplasty failure in diabetes, we examined serum and synovial fluid markers as well as collagen crosslinks in bone and cartilage of 20 patients (10 with diabetes, 10 controls without) undergoing this procedure. Hemoglobin A1c, body mass index, bone alkaline phosphatase, leptin, osteocalcin, and pyridinium were analyzed along with tissue content of the crosslinks hydroxylysylpyridinoline, lysylpyridinoline, and pentosidine. Pentosidine levels in tissue specimens from diabetic subjects were higher than in control subjects. Osteocalcin levels negatively correlated with hydroxylysylpyridinoline levels in cartilage. Osteocalcin levels also negatively correlated with pentosidine levels in cartilage, but only in subjects with diabetes. This study suggests potential metabolic mechanisms for arthroplasty failure in patients with diabetes.

Relationship of serum relaxin to generalized and trapezial-metacarpal joint laxity.

PURPOSE The reproductive hormone relaxin acts to loosen pelvic ligaments in preparation for childbirth and is thought to be a mediator of joint laxity. The purpose of this study was to evaluate the correlation of serum relaxin with radiographic laxity at the trapezial-metacarpal joint and with generalized joint laxity. METHODS We enrolled 289 healthy subjects prospectively. Participants completed a demographic questionnaire and were examined for generalized joint hypermobility using the Beighton-Horan scale. Stress radiographs of the trapezial-metacarpal joint were obtained in 163 subjects (56%). Blood samples were collected, and serum relaxin was measured for 287 subjects using enzyme-linked immunosorbent assay for human relaxin-2. RESULTS The mean serum relaxin level among all subjects was 1.84 pg/mL (range, 0-45.25 pg/mL). Relaxin was not detectable in 166 of 287 samples, whereas the mean serum relaxin level among the 121 subjects with a detectable relaxin level (of 287 total relaxin samples) was 4.37 pg/mL (range, 0.46-45.25 pg/mL). Mean trapezial-metacarpal subluxation ratio scores were higher among those with a detectable relaxin level compared to those without a detectable relaxin level (0.34 vs 0.30 pg/mL). The average Beighton-Horan laxity score was 1.8 (range, 0-9). There was no correlation between generalized joint laxity measures and serum relaxin levels. CONCLUSIONS In a large volunteer population, we demonstrated a relationship between circulating relaxin and trapezial-metacarpal joint laxity. However, we were unable to show a direct link between serum relaxin and generalized joint laxity. TYPE OF STUDY/LEVEL OF EVIDENCE Prognostic II.