Syrbactin-class dual constitutive- and immuno-proteasome inhibitor TIR-199 impedes myeloma-mediated bone degeneration in vivo.

Proteasome-addicted neoplastic malignancies present a considerable refractory and relapsed phenotype with patients exhibiting drug resistance and high mortality rates. To counter this global problem, novel proteasome-based therapies are being developed. In the current study, we extensively characterize TIR-199, a syrbactin-class proteasome inhibitor derived from a plant virulence factor of bacterium Pseudomonas syringae pv syringae. We report that TIR-199 is a potent constitutive and immunoproteasome inhibitor, capable of inducing cell death in multiple myeloma, triple-negative breast cancer, (TNBC) and non-small cell lung cancer lines. TIR-199 also effectively inhibits the proteasome in primary myeloma cells of patients, and bypasses the PSMB5 A49T+A50V bortezomib-resistant mutant. TIR-199 treatment leads to accumulation of canonical proteasome substrates in cells, it is specific, and does not inhibit 50 other enzymes tested in vitro. The drug exhibits synergistic cytotoxicity in combination with proteasome-activating kinase DYRK2 inhibitor LDN192960. Furthermore, low-doses of TIR-199 exhibits in vivo activity by delaying myeloma-mediated bone degeneration in a mouse xenograft model. Together, our data indicates that proteasome inhibitor TIR-199 could indeed be a promising next-generation drug within the repertoire of proteasome-based therapeutics.

Effects of an articular cartilage lubrication with a viscosupplement in vitro and in vivo following osteochondral fractures in horses.

OBJECTIVE: To assess whether the combination of hyaluronan, sodium chondroitin sulfate, and N-acetyl-d-glucosamine (HCSG) lubricates articular cartilage in vitro and modulates joint lubrication in vivo. ANIMALS: 16 healthy adult horses. PROCEDURES: The effects of HCSG injections on SF lubricant properties and joint health, immediately after injury and 2 weeks later, were analyzed by use an equine osteochondral fracture model of post-traumatic osteoarthritis (OA). Middle carpal joints of adult horses were randomly assigned to 1 of 4 surgical treatment groups as follows: normal nonsurgical group (n = 8), normal sham-surgical group (8), OA-induced surgical group with HCSG injection (8), or OA-induced surgical group with saline (0.9% NaCl) solution injection (8). Synovial fluid was aspirated periodically and analyzed for boundary lubrication function and lubricant molecules. At 17 days, joints were screened for gross pathological changes. RESULTS: Induction of OA led to an impairment of SF lubrication function and diminished hyaluronan concentration in a time-dependent manner following surgery, with HCSG injection lessening these effects. Certain friction coefficients approached those of unaffected normal equine SF. Induction of OA also caused synovial hemorrhage at 17 days, which was lower in joints treated with HCSG. CONCLUSIONS AND CLINICAL RELEVANCE: After induction of OA, equine SF lubricant function was impaired. Hyaluronan-sodium chondroitin sulfate-N-acetyl-d-glucosamine injection restored lubricant properties at certain time points and reduced pathological joint changes.

Matrix Rigidity Controls Epithelial-Mesenchymal Plasticity and Tumor Metastasis via a Mechanoresponsive EPHA2/LYN Complex.

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis in vivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.

Protein kinase G1 regulates bone regeneration and rescues diabetic fracture healing.

Bone fractures are a major cause of morbidity and mortality, particularly in patients with diabetes, who have a high incidence of fractures and exhibit poor fracture healing. Coordinated expression of osteoblast-derived vascular endothelial growth factor (VEGF) and bone morphogenic proteins (BMPs) is essential for fracture repair. The NO/cGMP/protein kinase G (PKG) signaling pathway mediates osteoblast responses to estrogens and mechanical stimulation, but the pathway's role in bone regeneration is unknown. Here, we used a mouse cortical-defect model to simulate bone fractures and studied osteoblast-specific PKG1-knockout and diabetic mice. The knockout mice had normal bone microarchitecture but after injury exhibited poor bone regeneration, with decreased osteoblasts, collagen deposition, and microvessels in the bone defect area. Primary osteoblasts and tibiae from the knockout mice expressed low amounts of Vegfa and Bmp2/4 mRNAs, and PKG1 was required for cGMP-stimulated expression of these genes. Diabetic mice also demonstrated low Vegfa and Bmp2/4 expression in bone and impaired bone regeneration after injury; notably, the cGMP-elevating agent cinaciguat restored Vegfa and BMP2/4 expression and full bone healing. We conclude that PKG1 is a key orchestrator of VEGF and BMP signaling during bone regeneration and propose pharmacological PKG activation as a novel therapeutic approach to enhance fracture healing.

Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.

Combinatorial targeting of cancer bone metastasis using mRNA engineered stem cells.

BACKGROUND: Bone metastases are common and devastating to cancer patients. Existing treatments do not specifically target the disease sites and are therefore ineffective and systemically toxic. Here we present a new strategy to treat bone metastasis by targeting both the cancer cells ("the seed"), and their surrounding niche ("the soil"), using stem cells engineered to home to the bone metastatic niche and to maximise local delivery of multiple therapeutic factors. METHODS: We used mesenchymal stem cells engineered using mRNA to simultaneously express P-selectin glycoprotein ligand-1 (PSGL-1)/Sialyl-Lewis X (SLEX) (homing factors), and modified versions of cytosine deaminase (CD) and osteoprotegerin (OPG) (therapeutic factors) to target and treat breast cancer bone metastases in two mouse models, a xenograft intratibial model and a syngeneic model of spontaneous bone metastasis. FINDINGS: We first confirmed that MSC engineered using mRNA produced functional proteins (PSGL-1/SLEX, CD and OPG) using various in vitro assays. We then demonstrated that mRNA-engineered MSC exhibit enhanced homing to the bone metastatic niche likely through interactions between PSGL-1/SLEX and P-selectin expressed on tumour vasculature. In both the xenograft intratibial model and syngeneic model of spontaneous bone metastasis, engineered MSC can effectively kill tumour cells and preserve bone integrity. The engineered MSC also exhibited minimal toxicity in vivo, compared to its non-targeted chemotherapy counterpart (5-fluorouracil). INTERPRETATION: Our combinatorial targeting of both the cancer cells and the niche represents a simple, safe and effective way to treat metastatic bone diseases, otherwise difficult to manage with existing strategies. It can also be applied to other cell types (e.g., T cells) and cargos (e.g., genome editing components) to treat a broad range of cancer and other complex diseases. FUND: National Institutes of Health, National Cancer Institute of the National Institutes of Health, Department of Defense, California Institute of Regenerative Medicine, National Science Foundation, Baylx Inc., and Fondation ARC pour la recherche sur le cancer.

ISSLS PRIZE IN BASIC SCIENCE 2018: Growth differentiation factor-6 attenuated pro-inflammatory molecular changes in the rabbit anular-puncture model and degenerated disc-induced pain generation in the rat xenograft radiculopathy model.

PURPOSE: To elucidate the effects of growth differentiation factor-6 (GDF6) on: (i) gene expression of inflammatory/pain-related molecules and structural integrity in the rabbit intervertebral disc (IVD) degeneration model, and (ii) sensory dysfunction and changes in pain-marker expression in dorsal nerve ganglia (DRGs) in the rat xenograft radiculopathy model. METHODS: Forty-six adolescent rabbits received anular-puncture in two non-consecutive lumbar IVDs. Four weeks later, phosphate-buffered saline (PBS) or GDF6 (1, 10 or 100 µg) was injected into the nucleus pulposus (NP) of punctured discs and followed for 4 weeks for gene expression analysis and 12 weeks for structural analyses. For pain assessment, eight rabbits were sacrificed at 4 weeks post-injection and NP tissues of injected discs were transplanted onto L5 DRGs of 16 nude rats to examine mechanical allodynia. The rat DRGs were analyzed immunohistochemically. RESULTS: In GDF6-treated rabbit NPs, gene expressions of interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, prostaglandin-endoperoxide synthase 2, and nerve growth factor were significantly lower than those in the PBS group. GDF6 injections resulted in partial restoration of disc height and improvement of MRI disc degeneration grades with statistical significance in rabbit structural analyses. Allodynia induced by xenograft transplantation of rabbit degenerated NPs onto rat DRGs was significantly reduced by GDF6 injection. Staining intensities for ionized calcium-binding adaptor molecule-1 and calcitonin gene-related peptide in rat DRGs of the GDF6 group were significantly lower than those of the PBS group. CONCLUSION: GDF6 injection may change the pathological status of degenerative discs and attenuate degenerated IVD-induced pain.

A Single-Blind Study Evaluating the Efficacy of Gold Nanoparticle Photothermal-Assisted Liposuction in an Ex Vivo Human Tissue Model.

BACKGROUND: Liposuction is one of the most performed cosmetic surgery procedures. In a previously reported study, gold-nanoparticle (GNP) laser-assisted liposuction (NanoLipo) was shown to improve procedure parameters and outcomes in a porcine model. OBJECTIVES: An ex vivo human liposuction model was developed to assess the ease, efficacy, and outcomes of NanoLipo, and to further explore its mechanism of action in facilitating liposuction. METHODS: NanoLipo was compared to a control without GNPs in sets of fresh, nonperfused, anatomically symmetric, matched tissue specimens from 12 patients. A subset of three experiments was performed under single-blinded conditions. Intraoperative assessments included lipoaspirate volume, percentage of free oil, ease of removal, and temperature rise. Specimens were palpated, visualized for evenness, and graded with and without skin. Postoperative assessment included viability staining of the lipoaspirate and remaining tissues. Microcomputed tomography was used to assess the distribution of infused GNPs within the tissues. RESULTS: NanoLipo consistently removed more adipose tissue with more liberated triglycerides compared to control. NanoLipo specimens were smoother, thinner, and had fewer and smaller irregularities. Infused solutions preferentially distributed between fibrous membranes and fat pearls. After NanoLipo, selective structural-tissue disruptions, indicated by loss of metabolic activity, were observed. Thus, NanoLipo likely creates a bimodal mechanism of action whereby fat lobules are dislodged from surrounding fibro-connective tissue, while lipolysis is simultaneously induced. CONCLUSIONS: NanoLipo showed many advantages compared to control under blinded and nonblinded conditions. This technology may be promising in facilitating fat removal.

Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes.

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat-an NO-independent activator of oxidized guanylate cyclase-increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

A Novel, Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice.

Most US Food and Drug Administration (FDA)-approved treatments for osteoporosis target osteoclastic bone resorption. Only PTH derivatives improve bone formation, but they have drawbacks, and novel bone-anabolic agents are needed. Nitrates, which generate NO, improved BMD in estrogen-deficient rats and may improve bone formation markers and BMD in postmenopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long-term use. Here we studied nitrosyl-cobinamide (NO-Cbi), a novel, direct NO-releasing agent, in a mouse model of estrogen deficiency-induced osteoporosis. In murine primary osteoblasts, NO-Cbi increased intracellular cGMP, Wnt/ß-catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in intact and ovariectomized (OVX) female C57Bl/6 mice, NO-Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and in OVX mice, it prevented osteocyte apoptosis. NO-Cbi reduced osteoclasts in intact mice and prevented the known increase in osteoclasts in OVX mice, partially through a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and partially through direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. The positive NO effects in osteoblasts were mediated by cGMP/protein kinase G (PKG), but some of the osteoclast-inhibitory effects appeared to be cGMP-independent. NO-Cbi increased trabecular bone mass in both intact and OVX mice, consistent with its in vitro effects on osteoblasts and osteoclasts. NO-Cbi is a novel direct NO-releasing agent that, in contrast to nitrates, does not generate oxygen radicals, and combines anabolic and antiresorptive effects in bone, making it an excellent candidate for treating osteoporosis. © 2016 American Society for Bone and Mineral Research.

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pi t1 Double-Knockout Mice.

We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte-derived and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of inorganic phosphate (Pi ) and phosphate transporter-mediated influx of Pi . To test this hypothesis, we generated mice with col2a1-driven Cre-mediated ablation of Slc20a1, hereafter referred to as Pi t1, alone or in combination with a Phospho1 gene deletion. Pi t1(col2/col2) mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1(-/-) ; Pi t1(col2/col2) ] double knockout mice that were more pronounced than those observed in the Phospho1(-/-) mice. Histological analysis of [Phospho1(-/-) ; Pi t1(col2/col2) ] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1(-/-) ; Pi t1(col2/col2) ] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1(-/-) mice. Using atomic force microscopy we found that ∼80% of [Phospho1(-/-) ; Pi t1(col2/col2) ] MVs were devoid of mineral in comparison to ∼50% for the Phospho1(-/-) MVs and ∼25% for the WT and Pi t1(col2/col2) MVs. We also found a significant decrease in the number of MVs produced by both Phospho1(-/-) and [Phospho1(-/-) ; Pi t1(col2/col2) ] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as Pi T-1, in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. © 2016 American Society for Bone and Mineral Research.

Addition of Mesenchymal Stem Cells to Autologous Platelet-Enhanced Fibrin Scaffolds in Chondral Defects: Does It Enhance Repair?

BACKGROUND: The chondrogenic potential of culture-expanded bone-marrow-derived mesenchymal stem cells (BMDMSCs) is well described. Numerous studies have also shown enhanced repair when BMDMSCs, scaffolds, and growth factors are placed into chondral defects. Platelets provide a rich milieu of growth factors and, along with fibrin, are readily available for clinical use. The objective of this study was to determine if the addition of BMDMSCs to an autologous platelet-enriched fibrin (APEF) scaffold enhances chondral repair compared with APEF alone. METHODS: A 15-mm-diameter full-thickness chondral defect was created on the lateral trochlear ridge of both stifle joints of twelve adult horses. In each animal, one defect was randomly assigned to receive APEF+BMDMSCs and the contralateral defect received APEF alone. Repair tissues were evaluated one year later with arthroscopy, histological examination, magnetic resonance imaging (MRI), micro-computed tomography (micro-CT), and biomechanical testing. RESULTS: The arthroscopic findings, MRI T2 map, histological scores, structural stiffness, and material stiffness were similar (p > 0.05) between the APEF and APEF+BMDMSC-treated repairs at one year. Ectopic bone was observed within the repair tissue in four of twelve APEF+BMDMSC-treated defects. Defects repaired with APEF alone had less trabecular bone edema (as seen on MRI) compared with defects repaired with APEF+BMDMSCs. Micro-CT analysis showed thinner repair tissue in defects repaired with APEF+BMDMSCs than in those treated with APEF alone (p < 0.05). CONCLUSIONS: APEF alone resulted in thicker repair tissue than was seen with APEF+BMDMSCs. The addition of BMDMSCs to APEF did not enhance cartilage repair and stimulated bone formation in some cartilage defects. CLINICAL RELEVANCE: APEF supported repair of critical-size full-thickness chondral defects in horses, which was not improved by the addition of BMDMSCs. This work supports further investigation to determine whether APEF enhances cartilage repair in humans.

Hyaluronan concentration and size distribution in human knee synovial fluid: variations with age and cartilage degeneration.

BACKGROUND: One potential mechanism for early superficial cartilage wear in normal joints is alteration of the lubricant content and quality of synovial fluid. The purpose of this study was to determine if the concentration and quality of the lubricant, hyaluronan, in synovial fluid: (1) was similar in left and right knees; (2) exhibited similar age-associated trends, whether collected postmortem or antemortem; and (3) varied with age and grade of joint degeneration. METHODS: Human synovial fluid of donors (23-91 years) without osteoarthritis was analyzed for the concentrations of protein, hyaluronan, and hyaluronan in the molecular weight ranges of 2.5-7 MDa, 1-2.5 MDa, 0.5-1 MDa, and 0.03-0.5 MDa. Similarity of data between left and right knees was assessed by reduced major axis regression, paired t-test, and Bland-Altman analysis. The effect of antemortem versus postmortem collection on biochemical properties was assessed for age-matched samples by unpaired t-test. The relationships between age, joint grade, and each biochemical component were assessed by regression analysis. RESULTS: Joint grade and the concentrations of protein, hyaluronan, and hyaluronan in the molecular weight ranges of 2.5-7 MDa, 1-2.5 MDa, and 0.5-1 MDa in human synovial fluid showed good agreement between left and right knees and were similar between age-matched patient and cadaver knee joints. There was an age-associated decrease in overall joint grade (-15 %/decade) and concentrations of hyaluronan (-10.5 %/decade), and hyaluronan in the molecular weight ranges of 2.5-7 MDa (-9.4 %/decade), 1-2.5 MDa (-11.3 %/decade), 0.5-1 MDa (-12.5 %/decade), and 0.03-0.5 MDa (-13.0 %/decade). Hyaluronan concentration and quality was more strongly associated with age than with joint grade. CONCLUSIONS: The age-related increase in cartilage wear in non-osteoarthritic joints may be related to the altered hyaluronan content and quality of synovial fluid.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer.

OBJECTIVE: Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined. METHODS: Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2-/-γc-/- male mice. RESULTS: Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft. CONCLUSION: PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.

Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway.

Matrix stiffness potently regulates cellular behaviour in various biological contexts. In breast tumours, the presence of dense clusters of collagen fibrils indicates increased matrix stiffness and correlates with poor survival. It is unclear how mechanical inputs are transduced into transcriptional outputs to drive tumour progression. Here we report that TWIST1 is an essential mechanomediator that promotes epithelial-mesenchymal transition (EMT) in response to increasing matrix stiffness. High matrix stiffness promotes nuclear translocation of TWIST1 by releasing TWIST1 from its cytoplasmic binding partner G3BP2. Loss of G3BP2 leads to constitutive TWIST1 nuclear localization and synergizes with increasing matrix stiffness to induce EMT and promote tumour invasion and metastasis. In human breast tumours, collagen fibre alignment, a marker of increasing matrix stiffness, and reduced expression of G3BP2 together predict poor survival. Our findings reveal a TWIST1-G3BP2 mechanotransduction pathway that responds to biomechanical signals from the tumour microenvironment to drive EMT, invasion and metastasis.

Synovial Fluid Lubricant Properties are Transiently Deficient after Arthroscopic Articular Cartilage Defect Repair with Platelet-Enriched Fibrin Alone and with Mesenchymal Stem Cells.

BACKGROUND: Following various types of naturally-occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan and proteoglycan-4. However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown. HYPOTHESIS: Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin+mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and proteoglycan-4 composition. STUDY DESIGN: Controlled Laboratory Study. METHODS: Equine synovial fluid was aspirated from normal joints at a pre-injury state (0 days) and at 10 days and 3 months following fibrin or fibrin+mesenchymal stem cell repair of full thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and proteoglycan-4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high-molecular weight hyaluronan to restore lubrication function. RESULTS: Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to pre-injury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (-64%) and shifted towards lower molecular weight forms, while proteoglycan-4 concentration was increased (+210%) in 10-day samples relative to pre-injury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high molecular weight hyaluronan. Addition of high molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0mg/ml restored kinetic friction to pre-injury levels. CONCLUSION: Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high molecular weight hyaluronan. CLINICAL RELEVANCE: Synovial fluid lubrication is deficient shortly following arthroscopic cartilage repair surgery, and supplementation with high molecular weight hyaluronan may be beneficial.

Development of a Comprehensive Osteochondral Allograft MRI Scoring System (OCAMRISS) with Histopathologic, Micro-Computed Tomography, and Biomechanical Validation.

OBJECTIVE: To describe and apply a semi-quantitative MRI scoring system for multi-feature analysis of cartilage defect repair in the knee by osteochondral allografts, and to correlate this scoring system with histopathologic, micro-computed tomography (µCT), and biomechanical reference standards using a goat repair model. DESIGN: Fourteen adult goats had two osteochondral allografts implanted into each knee: one in the medial femoral condyle (MFC) and one in the lateral trochlea (LT). At 12 months, goats were euthanized and MRI was performed. Two blinded radiologists independently rated nine primary features for each graft, including cartilage signal, fill, edge integration, surface congruity, calcified cartilage integrity, subchondral bone plate congruity, subchondral bone marrow signal, osseous integration, and presence of cystic changes. Four ancillary features of the joint were also evaluated, including opposing cartilage, meniscal tears, synovitis, and fat-pad scarring. Comparison was made with histological and µCT reference standards as well as biomechanical measures. Interobserver agreement and agreement with reference standards was assessed. Cohen's kappa, Spearman's correlation, and Kruskal-Wallis tests were used as appropriate. RESULTS: There was substantial agreement (κ>0.6, p<0.001) for each MRI feature and with comparison against reference standards, except for cartilage edge integration (κ=0.6). There was a strong positive correlation between MRI and reference standard scores (ρ=0.86, p<0.01). OCAMRISS was sensitive to differences in outcomes between the types of allografts. CONCLUSIONS: We have described a comprehensive MRI scoring system for osteochondral allografts and have validated this scoring system with histopathologic and µCT reference standards as well as biomechanical indentation testing.

Micro-computed tomography-based three-dimensional kinematic analysis during lateral bending for spinal fusion assessment in a rat posterolateral lumbar fusion model.

Rat posterolateral lumbar fusion (PLF) models have been used to assess the safety and effectiveness of new bone substitutes and osteoinductive growth factors using palpation, radiography, micro-computed tomography (µCT), and histology as standard methods to evaluate spinal fusion. Despite increased numbers of PLF studies involving alternative bone substitutes and growth factors, the quantitative assessment of treatment efficacy during spinal motion has been limited. The purpose of this study was to evaluate the effect of spinal fusion on lumbar spine segment stability during lateral bending using a µCT-based three-dimensional (3D) kinematic analysis in the rat PLF model. Fourteen athymic male rats underwent PLF surgery at L4/5 and received bone grafts harvested from the ilium and femurs of syngeneic rats (Isograft, n=7) or no graft (Sham, n=7). At 8 weeks after the PLF surgery, spinal fusion was assessed by manual palpation, plain radiography, µCT, and histology. To determine lumbar segmental motions at the operated level during lateral bending, 3D kinematic analysis was performed. The Isograft group, but not the Sham group, showed spinal fusion on manual palpation (6/7), solid fusion mass in radiographs (6/7), as well as bone bridging in µCT and histological images (5/7). Compared to the Sham group, the Isograft group revealed limited 3D lateral bending angular range of motion and lateral translation during lateral bending at the fused segment where disc height narrowing was observed. This µCT-based 3D kinematic analysis can provide a quantitative assessment of spinal fusion in a rat PLF model to complement current gold standard methods used for efficacy assessment of new therapeutic approaches.

Volume Expansion of Tissue Engineered Human Nasal Septal Cartilage.

IMPORTANCE: Cartilaginous craniofacial defects range in size and autologous cartilaginous tissue is preferred for repair of these defects. Therefore, it is important to have the ability to produce large size cartilaginous constructs for repair of cartilaginous abnormalities. OBJECTIVES: To produce autologous human septal neocartilage constructs substantially larger in size than previously produced constructsTo demonstrate that volume expanded neocartilage constructs possess comparable histological and biochemical properties to standard size constructsTo show that volume expanded neocartilage constructs retain similar biomechanical properties to standard size constructs. DESIGN: Prospective, basic science. SETTING: Laboratory. PARTICIPANTS: The study used remnant human septal specimens removed during routine surgery at the University of California, San Diego Medical Center or San Diego Veterans Affairs Medical Center. Cartilage from a total of 8 donors was collected. MAIN OUTCOMES MEASURED: Human septal chondrocytes from 8 donors were used to create 12mm and 24mm neocartilage constructs. These were cultured for a total of 10 weeks. Photo documentation, histological, biochemical, and biomechanical properties were measured and compared. RESULTS: The 24mm diameter constructs were qualitatively similar to the 12mm constructs. They possessed adequate strength and durability to be manually manipulated. Histological analysis of the constructs demonstrated similar staining patterns in standard and volume expanded constructs. Proliferation, as measured by DNA content, was similar in 24mm and 12mm constructs. Additionally, glycosaminoglycan (GAG) and total collagen content did not significantly differ between the two construct sizes. Biomechanical analysis of the 24mm and 12mm constructs demonstrated comparable compressive and tensile properties. CONCLUSION AND RELEVANCE: Volume expanded human septal neocartilage constructs are qualitatively and histologically similar to standard 12mm constructs. Biochemical and biomechanical analysis of the constructs demonstrated equivalent properties. This study shows that modification of existing protocols is not required to successfully produce neocartilage constructs in larger sizes for reconstruction of more substantial craniofacial defects. LEVEL OF EVIDENCE: NA.