Non-invasive electroarthrography measures cartilage in live horses and correlates to direct measurements of cartilage streaming potentials in weight bearing regions of equine metacarpophalangeal joints.

OBJECTIVE: To perform non-invasive Electroarthrography (EAG) on live horses and establish relationships between EAG and direct measurements of cartilage streaming potentials in weight bearing areas of the equine metacarpophalangeal joint. DESIGN: EAG was performed bilaterally on the metacarpophalangeal joints of live horses (n = 3). Separate experiments used metacarpophalangeal joint explants (n = 11) to measure EAG obtained during simulated loading followed by direct measurements of cartilage streaming potentials on joint surfaces using the Arthro-BST probe. Joints were assigned to relatively normal (n = 5) and mildly degraded (n = 6) groups based on histological scoring of Safranin-O/Fast Green stained sections. RESULTS: EAG, involving application of electrodes to skin surrounding the joint and repeated weight shifting, was well-tolerated in live horses. One pair of distal forelimbs were available for analogous ex vivo EAG testing and measurements were strongly correlated to in vivo EAG measurements obtained on the same joints (r = 0.804, p = 0.016, n = 8). Both indirect (EAG) and direct (Arthro-BST) measurements of cartilage streaming potentials distinguished between normal and mildly degraded cartilage with statistically significant differences at 5 of 6 and 4 of 6 electrodes during simulated standing and walking, respectively. Strong and moderate correlations for weight bearing regions on the dorsal phalanx and central metacarpus were detected during both standing and walking. At the metacarpus/sesamoid interface a moderate correlation occurred during walking. CONCLUSION: Non-invasive EAG was used successfully in a clinical scenario and correlated to direct measurements of streaming potentials in weight bearing cartilage. These data support the potential of EAG to contribute to the diagnosis and treatment of degenerative joint diseases.

Increased Osteoblast GαS Promotes Ossification by Suppressing Cartilage and Enhancing Callus Mineralization During Fracture Repair in Mice.

GαS, the stimulatory G protein α-subunit that raises intracellular cAMP levels by activating adenylyl cyclase, plays a vital role in bone development, maintenance, and remodeling. Previously, using transgenic mice overexpressing GαS in osteoblasts (GS-Tg), we demonstrated the influence of osteoblast GαS level on osteogenesis, bone turnover, and skeletal responses to hyperparathyroidism. To further investigate whether alterations in GαS levels affect endochondral bone repair, a postnatal bone regenerative process that recapitulates embryonic bone development, we performed stabilized tibial osteotomy in male GS-Tg mice at 8 weeks of age and examined the progression of fracture healing by micro-CT, histomorphometry, and gene expression analysis over a 4-week period. Bone fractures from GS-Tg mice exhibited diminished cartilage formation at the time of peak soft callus formation at 1 week post-fracture followed by significantly enhanced callus mineralization and new bone formation at 2 weeks post-fracture. The opposing effects on chondrogenesis and osteogenesis were validated by downregulation of chondrogenic markers and upregulation of osteogenic markers. Histomorphometric analysis at times of increased bone formation (2 and 3 weeks post-fracture) revealed excess fibroblast-like cells on newly formed woven bone surfaces and elevated osteocyte density in GS-Tg fractures. Coincident with enhanced callus mineralization and bone formation, GS-Tg mice showed elevated active ß-catenin and Wntless proteins in osteoblasts at 2 weeks post-fracture, further substantiated by increased mRNA encoding various canonical Wnts and Wnt target genes, suggesting elevated osteoblastic Wnt secretion and Wnt/ß-catenin signaling. The GS-Tg bony callus at 4 weeks post-fracture exhibited greater mineral density and decreased polar moment of inertia, resulting in improved material stiffness. These findings highlight that elevated GαS levels increase Wnt signaling, conferring an increased osteogenic differentiation potential at the expense of chondrogenic differentiation, resulting in improved mechanical integrity. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Irregular porous titanium enhances implant stability and bone ingrowth in an intra-articular ovine model.

Two commercially available porous coatings, Gription and Porocoat, were compared for the first time in a challenging intra-articular, weight-bearing, ovine model. Gription has evolved from Porocoat and has higher porosity, coefficient of friction, and microtextured topography, which are expected to enhance bone ingrowth. Cylindrical implants were press-fit into the weight-bearing regions of ovine femoral condyles and bone ingrowth and fixation strength evaluated 4, 8, and 16 weeks postoperatively. Biomechanical push-out tests were performed on lateral femoral condyles (LFCs) to evaluate the strength of the bone-implant interface. Bone ingrowth was assessed in medial femoral condyles (MFCs) as well as implants retrieved from LFCs following biomechanical testing using backscattered electron microscopy and histology. By 16 weeks, Gription-coated implants exhibited higher force (2455 ± 1362 vs. 1002 ± 1466 N; p = 0.046) and stress (12.60 ± 6.99 vs. 5.14 ± 7.53 MPa; p = 0.046) at failure, and trended towards higher stiffness (11,510 ± 7645 vs. 5010 ± 8374 N/mm; p = 0.061) and modulus of elasticity (591 ± 392 vs. 256 ± 431 MPa; p = 0.061). A strong, positive correlation was detected between bone ingrowth in LFC implants and failure force (r = 0.93, p < 10-13 ). By 16 weeks, bone ingrowth in Gription-coated implants in MFCs was 10.50 ± 6.31% compared to 5.88 ± 2.77% in Porocoat (p = 0.095). Observations of the bone-implant interface, made following push-out testing, showed more bony material consistently adhered to Gription compared to Porocoat at all three time points. Gription provided superior fixation strength and bone ingrowth by 16 weeks.

Non-invasive Electroarthrography Measures Load-Induced Cartilage Streaming Potentials via Electrodes Placed on Skin Surrounding an Articular Joint.

OBJECTIVE: We aimed to demonstrate that electroarthrography (EAG) measures streaming potentials originating in the cartilage extracellular matrix during load bearing through electrodes adhered to skin surrounding an articular joint. DESIGN: Equine metacarpophalangeal joints were subjected to simulated physiological loads while (1) replacing synovial fluid with immersion buffers of different electrolyte concentrations and (2) directly degrading cartilage with trypsin. RESULTS: An inverse relationship between ionic strength and EAG coefficient was detected. Compared to native synovial fluid, EAG coefficients increased (P < 0.05) for 5 of 6 electrodes immersed in 0.1X phosphate-buffered saline (PBS) (0.014 M NaCl), decreased (P < 0.05) for 4 of 6 electrodes in 1X PBS (0.14 M NaCl), and decreased (P < 0.05) for all 6 electrodes in 10X PBS (1.4 M NaCl). This relationship corresponds to similar studies where streaming potentials were directly measured on cartilage. EAG coefficients, obtained after trypsin degradation, were reduced (P < 0.05) in 6 of 8, and 7 of 8 electrodes, during simulated standing and walking, respectively. Trypsin degradation was confirmed by direct cartilage assessments. Streaming potentials, measured by directly contacting cartilage, indicated lower cartilage stiffness (P < 10-5). Unconfined compression data revealed reduced Em, representing proteoglycan matrix stiffness (P = 0.005), no change in Ef, representing collagen network stiffness (P = 0.15), and no change in permeability (P = 0.24). Trypsin depleted proteoglycan as observed by both dimethylmethylene blue assay (P = 0.0005) and safranin-O stained histological sections. CONCLUSION: These data show that non-invasive EAG detects streaming potentials produced by cartilage during joint compression and has potential to become a diagnostic tool capable of detecting early cartilage degeneration.

Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced ß-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146-/- mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate ß-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development.

RANKL coordinates multiple osteoclastogenic pathways by regulating expression of ubiquitin ligase RNF146.

Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of ß-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of ß-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases.

Calcium polyphosphate particulates for bone void filler applications.

This study investigates the characteristics of porous calcium polyphosphate particulates (CPPp) formed using two different processing treatments as bone void fillers in non- or minimally load-bearing sites. The two calcium polyphosphate particulate variants (grades) were formed using different annealing conditions during particulate preparation to yield either more slowly degrading calcium polyphosphate particulates (SD-CPPp) or faster degrading particulates (FD-CPPp) as suggested by a previous degradation study conducted in vitro (Hu et al., Submitted for publication 2016). The two CPPp grades were compared as bone void fillers in vivo by implanting particulates in defects created in rabbit femoral condyle sites (critical size defects). The SD-CPPp and FD-CPPp were implanted for 4- and 16-week periods. The in vivo study indicated a significant difference in amount of new bone formed in the prepared sites with SD-CPPp resulting in more new bone formation compared with FD-CPPp. The lower bone formation characteristic of the FD-CPPp was attributed to its faster degradation rate and resulting higher local concentration of released polyphosphate degradation products. The study results indicate the importance of processing conditions on preparing calcium polyphosphate particulates for potential use as bone void fillers in nonload-bearing sites. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 874-884, 2017.

Osteochondral Biopsy Analysis Demonstrates That BST-CarGel Treatment Improves Structural and Cellular Characteristics of Cartilage Repair Tissue Compared With Microfracture.

OBJECTIVE: The efficacy and safety of BST-CarGel, a chitosan-based medical device for cartilage repair, was compared with microfracture alone at 1 year during a multicenter randomized controlled trial (RCT) in the knee. The quality of repair tissue of osteochondral biopsies collected from a subset of patients was compared using blinded histological assessments. METHODS: The international RCT evaluated repair tissue quantity and quality by 3-dimensional quantitative magnetic resonance imaging as co-primary endpoints at 12 months. At an average of 13 months posttreatment, 21/41 BST-CarGel and 17/39 microfracture patients underwent elective second look arthroscopies as a tertiary endpoint, during which ICRS (International Cartilage Repair Society) macroscopic scoring was carried out, and osteochondral biopsies were collected. Stained histological sections were evaluated by blinded readers using ICRS I and II histological scoring systems. Collagen organization was evaluated using a polarized light microscopy score. RESULTS: BST-CarGel treatment resulted in significantly better ICRS macroscopic scores (P = 0.0002) compared with microfracture alone, indicating better filling, integration, and tissue appearance. Histologically, BST-CarGel resulted in a significant improvement of structural parameters-Surface Architecture (P = 0.007) and Surface/Superficial Assessment (P = 0.042)-as well as cellular parameters-Cell Viability (P = 0.006) and Cell Distribution (P = 0.032). No histological parameters were significantly better for the microfracture group. BST-CarGel treatment also resulted in a more organized repair tissue with collagen stratification more similar to native hyaline cartilage, as measured by polarized light microscopy scoring (P = 0.0003). CONCLUSION: Multiple and independent analyses in this biopsy substudy demonstrated that BST-CarGel treatment results in improved structural and cellular characteristics of repair tissue at 1 year posttreatment compared with microfracture alone, supporting previously reported results by quantitative magnetic resonance imaging.

Chondroinduction Is the Main Cartilage Repair Response to Microfracture and Microfracture With BST-CarGel: Results as Shown by ICRS-II Histological Scoring and a Novel Zonal Collagen Type Scoring Method of Human Clinical Biopsy Specimens.

BACKGROUND: Current cartilage repair histological scoring systems are unable to explain the relationship between collagen type II deposition and overall repair quality. PURPOSE/HYPOTHESIS: The purpose of this study was to develop a novel zonal collagen type (ZCT) 5-point scoring system to measure chondroinduction in human clinical biopsy specimens collected after marrow stimulation. The hypothesis was that the ZCT scores would correlate with the International Cartilage Repair Society-II (ICRS-II) overall histological repair assessment score and glycosaminoglycan (GAG) content. STUDY DESIGN: Descriptive laboratory study. METHODS: After optimizing safranin O staining for GAG and immunostaining for human collagen type II and type I (Col2 and Col1, respectively), serial sections from clinical osteochondral repair biopsy specimens (13 months after microfracture or microfracture with BST-CarGel; n = 39 patients) were stained and 3 blinded readers performed histomorphometry for percentage of staining, ICRS-II histological scoring, polarized light microscopy (PLM) scoring, and 5-point ZCT scoring based on tidemark morphology, zonal distribution of Col2 and Col1, and Col1 percentage stain. Because 1 biopsy specimen was missing bone, 38 biopsy specimens were evaluated for ICRS-II, PLM, and ZCT scores. RESULTS: Chondroinduction was identified in 21 biopsy specimens as a Col2 matrix fused to bone that spanned the deep-middle-superficial zones ("full-thickness hyaline repair"), deep-middle zones, or deep zone ("stalled hyaline") that was covered with a variable-thickness Col1-positive matrix, and was scored, respectively, as ZCT = 1 (n = 4 biopsy specimens), ZCT = 2 (n = 6) and ZCT = 3 (n = 11). Other biopsy specimens (n = 17) were fibrocartilage (n = 9; ZCT = 4), fibrous tissue (n = 4, ZCT = 5), or non-marrow derived (n = 4; ZCT = 0). Non-marrow derived tissue had a mean mature tidemark score of 84 out of 100 versus a regenerating tidemark score of 24 for all other biopsy specimens (P = .005). Both "stalled hyaline" repair and fibrocartilage had the same mean Col2 percentage stain; however, fibrocartilage was distinguished by heavy Col1 deposits in the deep zone, a 2-fold higher mean Col1 percentage stain (P = .001), and lower surface integrity (P = .03). ZCT scores correlated with GAG content and the ICRS-II overall assessment score, especially when combined with the PLM score for collagen organization (R = 0.82). Histological scores of the deep zone strongly predicted the ICRS-II overall assessment score (R = 0.99). CONCLUSION: The ICRS-II overall repair assessment score and GAG content correlated with the extent of Col2 deposition free of fibrosis in the deep/middle zone rather than bulk accumulation of Col2. CLINICAL RELEVANCE: Biopsy tissue from the BST-CarGel randomized clinical trial (microfracture without and with BST-CarGel, as treatment groups were not unblinded) showed regenerated tissue consistent with a chondroinduction mechanism in at least half of the treated lesions.

Effects of refrigeration and freezing on the electromechanical and biomechanical properties of articular cartilage.

In vitro electromechanical and biomechanical testing of articular cartilage provide critical information about the structure and function of this tissue. Difficulties obtaining fresh tissue and lengthy experimental testing procedures often necessitate a storage protocol, which may adversely affect the functional properties of cartilage. The effects of storage at either 4°C for periods of 6 days and 12 days, or during a single freeze-thaw cycle at -20°C were examined in young bovine cartilage. Non-destructive electromechanical measurements and unconfined compression testing on 3 mm diameter disks were used to assess cartilage properties, including the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Cartilage disks were also examined histologically. Compared with controls, significant decreases in SPI (to 32.3±5.5% of control values, p<0.001), Ef (to 31.3±41.3% [corrected] of control values, p=0.046), Em (to 6.4±8.5% of control values, p<0.0001), and an increase in k (to 2676.7±2562.0% of control values, p=0.004) were observed at day 12 of refrigeration at 4°C, but no significant changes were detected at day 6. A trend toward detecting a decrease in SPI (to 94.2±6.2% of control values, p=0.083) was identified following a single freeze-thaw cycle, but no detectable changes were observed for any biomechanical parameters. All numbers are mean±95% confidence interval. These results indicate that fresh cartilage can be stored in a humid chamber at 4°C for a maximum of 6 days with no detrimental effects to cartilage electromechanical and biomechanical properties, while one freeze-thaw cycle produces minimal deterioration of biomechanical and electromechanical properties. A comparison to literature suggested that particular attention should be paid to the manner in which specimens are thawed after freezing, specifically by minimizing thawing time at higher temperatures.

Osteochondral graft fixation using a bioresorbable bone cement.

Multiple osteochondral grafts can be used to resurface large joint defects in both humans and horses. In humans, immediate postoperative weight bearing can be prevented, however in the equine, it is unavoidable. Early weight bearing can create detrimental graft micromotion. The aim of this study was to investigate the role of a bioresorbable cement in improving the initial stability of multiple osteochondral graft repairs of large subchondral cystic lesions in the horse. Configurations employed for filling a 20mm diameter cylindrical defect included: (A) twelve 4.5mm diameter grafts with cement, (B) five 6.5mm diameter grafts with cement, (C) four each of 4.5mm and 6.5mm grafts with cement and (D) cement only. Intact bone slices (E) were also tested. Push-out tests were used to quantify construct to host sidewall interface fixation. Configuration (A) proved clinically impractical (n=3). Configurations (B) (n=6), and (C) (n=4) had statistically similar interface stiffnesses and failure stresses (43+/-8 and 30+/-12 MPa and 0.96+/-0.1 and 1.2+/-0.3 Mpa, respectively) suggesting that they are equally susceptible to interface movement in the immediate postoperative period. By way of comparison, defects filled only with cement had an average stiffness of 53+/-7MPa and failure stress of 1.8+/-0.3 MPa (n=6) while the intact femoral condyle demonstrated a stiffness of 108+/-7 MPa and failure stress of 18+/-0.4 MPa (n=6). Cement augmentation improved immediate postoperative stability of multiple osteochondral graft constructs over uncemented constructs, although in all cases the observed moduli of elasticity and yield stress values were lower than those observed for cement only and intact bone test specimens. (all numbers are mean+/-SEM).