D. Rick Sumner

The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor

Gene therapy using non-viral vectors that are safe and efficient in transfecting target cells is an effective approach to overcome the shortcomings of protein delivery of growth factors. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold to deliver polyethylenimine (PEI)-plasmid DNA (pDNA) [encoding platelet derived growth factor-B (PDGF-B)] complexes. The PEI-pPDGF-B complexes were fabricated at amine (N) to phosphate (P) ratio of 10 and characterized for size, surface charge, and in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. The complexes were ~100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. Confocal microscopy revealed significant proliferation of BMSCs on complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the complex-activated scaffolds following 4 weeks of implantation (14- and 44-fold higher) when compared to empty defects or empty scaffolds, respectively. Together, these findings suggest that non-viral PDGF-B gene-activated scaffolds are effective for bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.

Co-Cr-Mo alloy particles induce tumor necrosis factor alpha production in MLO-Y4 osteocytes: A role for osteocytes in particle-induced inflammation

Wear debris-induced osteolysis is purportedly the limiting problem affecting the long term results of joint arthroplasty. Pathogenic effects of wear debris in peri-implant cells such as macrophages, osteoblasts and osteoclasts have been well studied. In contrast, the effects of wear debris on osteocytes, which make up over 90% of all bone cells, remain unknown. We hypothesized that metal implant debris can induce the pro-inflammatory response in osteocytes. This study demonstrated the effects of cobalt-chromium-molybdenum alloy (Co-Cr-Mo) particles on a well-characterized MLO-Y4 osteocyte cell line. Co-Cr-Mo alloy particle treatment significantly (p<0.05) up-regulated tumor necrosis factor alpha (TNFalpha) gene expression after 3 and 6 h and TNFalpha protein production after 24 h, but down-regulated interleukin-6 (IL-6) gene expression after 6 h. Co-Cr-Mo alloy particle treatment also induced osteocyte apoptosis after 24 h. This apoptotic effect was partially (40%) dependent on TNFalpha. Therefore, our results suggest that osteocytes play a role in particle-induced inflammation and bone resorption following total joint arthroplasty by inducing pro-inflammatory cytokines and inducing osteocyte apoptosis.

Bone matrix quality after sclerostin antibody treatment.

Sclerostin antibody (Scl‐Ab) is a novel bone‐forming agent that is currently undergoing preclinical and clinical testing. Scl‐Ab treatment is known to dramatically increase bone mass, but little is known about the quality of the bone formed during treatment. In the current study, global mineralization of bone matrix in rats and nonhuman primates treated with vehicle or Scl‐Ab was assayed by backscattered scanning electron microscopy (bSEM) to quantify the bone mineral density distribution (BMDD). Additionally, fluorochrome labeling allowed tissue age–specific measurements to be made in the primate model with Fourier‐transform infrared microspectroscopy to determine the kinetics of mineralization, carbonate substitution, crystallinity, and collagen cross‐linking. Despite up to 54% increases in the bone volume after Scl‐Ab treatment, the mean global mineralization of trabecular and cortical bone was unaffected in both animal models investigated. However, there were two subtle changes in the BMDD after Scl‐Ab treatment in the primate trabecular bone, including an increase in the number of pixels with a low mineralization value (Z5) and a decrease in the standard deviation of the distribution. Tissue age–specific measurements in the primate model showed that Scl‐Ab treatment did not affect the mineral‐to‐matrix ratio, crystallinity, or collagen cross‐linking in the endocortical, intracortical, or trabecular compartments. Scl‐Ab treatment was associated with a nonsignificant trend toward accelerated mineralization intracortically and a nearly 10% increase in carbonate substitution for tissue older than 2 weeks in the trabecular compartment (p < 0.001). These findings suggest that Scl‐Ab treatment does not negatively impact bone matrix quality.

Temporal Gene Expression Profiling during Rat Femoral Marrow Ablation-Induced Intramembranous Bone Regeneration

Enhanced understanding of differential gene expression and biological pathways associated with distinct phases of intramembranous bone regeneration following femoral marrow ablation surgery will improve future advancements regarding osseointegration of joint replacement implants, biomaterials design, and bone tissue engineering. A rat femoral marrow ablation model was performed and genome-wide microarray data were obtained from samples at 1, 3, 5, 7, 10, 14, 28, and 56 days post-ablation, with intact bones serving as controls at Day 0. Bayesian model-based clustering produced eight distinct groups amongst 9,062 significant gene probe sets based on similar temporal expression profiles, which were further categorized into three major temporal classes of increased, variable, and decreased expression. Osteoblastic- and osteoclastic-associated genes were found to be significantly expressed within the increased expression groups. Chondrogenesis was not detected histologically. Adipogenic marker genes were found within variable/decreased expression groups, emphasizing that adipogenesis was inhibited during osteogenesis. Differential biological processes and pathways associated with each major temporal group were identified, and significantly expressed genes involved were visually represented by heat maps. It was determined that the increased expression group exclusively contains genes involved in pathways for matrix metalloproteinases (MMPs), Wnt signaling, TGF-β signaling, and inflammatory pathways. Only the variable expression group contains genes associated with glycolysis and gluconeogenesis, the notch signaling pathway, natural killer cell mediated cytotoxicity, and the B cell receptor signaling pathway. The decreased group exclusively consists of genes involved in heme biosynthesis, the p53 signaling pathway, and the hematopoietic cell lineage. Significant biological pathways and transcription factors expressed at each time point post-ablation were also identified. These data present the first temporal gene expression profiling analysis of the rat genome during intramembranous bone regeneration induced by femoral marrow ablation.

The use of intra‐articular Na‐hyaluronate as a potential chondroprotective device in experimentally induced acute articular cartilage injury and repair in rabbits

Objective: This study examined if viscosupplementation from intra‐articular administration of a commercially available form of hyaluronan (HA) could promote the restoration of proteoglycan (PG) depletion induced by chymopapain and then if the repair could be maintained once HA treatment was discontinued.

Chemically modified RNA activated matrices enhance bone regeneration

There exists a dire need for improved therapeutics to achieve predictable bone regeneration. Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is a promising approach to overcoming the drawbacks of protein delivery of growth factors. Here, we investigated the transfection efficiency, cytotoxicity, osteogenic potential and in vivo bone regenerative capacity of chemically modified ribonucleic acid (cmRNA) (encoding BMP-2) complexed with polyethylenimine (PEI) and made comparisons with PEI complexed with conventional plasmid DNA (encoding BMP-2). The polyplexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficiency using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these polyplexes was validated by determining the expression of bone-specific genes, osteocalcin and alkaline phosphatase as well as through the detection of bone matrix deposition. Using a calvarial bone defect model in rats, it was shown that PEI-cmRNA (encoding BMP-2)-activated matrices promoted significantly enhanced bone regeneration compared to PEI-plasmid DNA (BMP-2)-activated matrices. Our proof of concept study suggests that scaffolds loaded with non-viral vectors harboring cmRNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

Incorporation of copper into chitosan scaffolds promotes bone regeneration in rat calvarial defects

The objective of this study was to investigate the effects of a copper loaded chitosan scaffold on bone regeneration in critical-sized calvarial defects in rats. Chitosan scaffolds and copper-chitosan scaffolds were fabricated and characterized by scanning electron microscopy (SEM). Chitosan and copper-chitosan scaffolds were implanted into 5 mm diameter critical-sized calvarial defects in Fisher 344 male rats. Empty defects (no scaffolds) were included as a control. After 4 weeks, the rats were sacrificed for microcomputed tomography (micro-CT) and histological analysis of new bone tissue development. Microscopy images revealed the uniformly porous structure of chitosan and copper-chitosan scaffolds. Significant bone regeneration was noted in the defects treated with copper-chitosan scaffolds when evaluated using micro-CT and histological analysis, when compared with other groups tested. On analysis of the micro-CT scans, an eleven-fold and a two-fold increase in the new bone volume/total volume (BV/TV) % was found in defects treated with the copper-chitosan scaffolds, when compared to empty defects and chitosan scaffolds, respectively. This study demonstrated the suitability of copper-crosslinked chitosan scaffolds for bone tissue engineering and provides the first evidence that inclusion of copper ions in scaffolds can enhance tissue regeneration.

Are There Biological Markers for Wear or Corrosion? A Systematic Review

BackgroundIdentification of biomarkers associated with wear and tribocorrosion in joint arthroplasty would be helpful to enhance early detection of aseptic loosening and/or osteolysis and to improve understanding of disease progression. There have been several new reports since the last systematic review (which covered research through mid-2008) justifying a new assessment.Questions/purposesWe sought to determine which biomarkers have the most promise for early diagnosis and monitoring of aseptic loosening and/or osteolysis related to wear or corrosion in total joint arthroplasty.MethodsWe performed a systematic review using MEDLINE and EMBASE databases, covering the period through December 2013, and identified 1050 articles. We restricted the definition of biomarker to biomolecules and imaging parameters useful for diagnosis and monitoring of disease progression, only including articles in English. We chose 65 articles for full review, including 44 from the original search and 21 from subsequent hand searches. We used the 22 articles in which patients with total joint arthroplasty who had aseptic loosening and/or periimplant osteolysis unrelated to sepsis had been compared with patients with total joint arthroplasty with stable implants. There were 90 comparisons of these two patient populations involving 35 different biomarkers.ResultsDiagnostic accuracy was assessed in nine of the 90 comparisons with the highest accuracy found for tartrate-resistant acid phosphatase 5b (0.96), although a separate comparison for this biomarker found a lower accuracy (0.76). Accuracy of > 0.80 was also found for crosslinked n-telopeptide of type I collagen, osteoprotegerin, and deoxypyridinoline. The most studied markers, tumor necrosis factor-α and interleukin-1β, were found to differ in the affected and control groups in < 30% of the comparisons. Thirty of the 35 biomarkers were studied in four or fewer separate comparisons with nearly half of the biomarkers (17) studied in only one comparison. Many of the comparisons were not able to eliminate a number of confounding variables, and there was only one prospective study.ConclusionsCurrently, there are no validated biomarkers for early diagnosis and monitoring of the biological sequelae of wear or tribocorrosion, although there are some promising leads, including markers of bone turnover.

Assessment of glenoid chondral healing: comparison of microfracture to autologous matrix-induced chondrogenesis in a novel rabbit shoulder model.

BACKGROUND Management of glenohumeral arthrosis in young patients is a considerable challenge, with a growing need for non-arthroplasty alternatives. The objectives of this study were to develop an animal model to study glenoid cartilage repair and to compare surgical repair strategies to promote glenoid chondral healing. METHODS Forty-five rabbits underwent unilateral removal of the entire glenoid articular surface and were divided into 3 groups--untreated defect (UD), microfracture (MFx), and MFx plus type I/III collagen scaffold (autologous matrix-induced chondrogenesis [AMIC])--for the evaluation of healing at 8 weeks (12 rabbits) and 32 weeks (33 rabbits) after injury. Contralateral shoulders served as unoperated controls. Tissue assessments included 11.7-T magnetic resonance imaging (long-term healing group only), equilibrium partitioning of an ionic contrast agent via micro-computed tomography (EPIC-μCT), and histologic investigation (grades on International Cartilage Repair Society II scoring system). RESULTS At 8 weeks, x-ray attenuation, thickness, and volume did not differ by treatment group. At 32 weeks, the T2 index (ratio of T2 values of healing to intact glenoids) was significantly lower for the MFx group relative to the AMIC group (P = .01) whereas the T1ρ index was significantly lower for AMIC relative to MFx (P = .01). The micro-computed tomography-derived repair tissue volume was significantly higher for MFx than for UD. Histologic investigation generally suggested inferior healing in the AMIC and UD groups relative to the MFx group, which exhibited improvements in both integration of repair tissue with subchondral bone and tidemark formation over time. DISCUSSION Improvements conferred by AMIC were limited to magnetic resonance imaging outcomes, whereas MFx appeared to promote increased fibrous tissue deposition via micro-computed tomography and more hyaline-like repair histologically. The findings from this novel model suggest that MFx promotes biologic resurfacing of full-thickness glenoid articular injury.

HBM Mice Have Altered Bone Matrix Composition and Improved Material Toughness

The G171V mutation in the low-density lipoprotein receptor-related protein 5 (LRP5) leads to a high bone mass (HBM) phenotype. Studies using HBM transgenic mouse models have consistently found increased bone mass and whole-bone strength, but little attention has been paid to the composition of the bone matrix. The current study sought to determine if the cortical bone matrix composition differs in HBM and wild-type mice and to determine how much of the variance in bone material properties is explained by variance in matrix composition. Consistent with previous studies, HBM mice had greater cortical area, moment of inertia, ultimate force, bending stiffness, and energy to failure than wild-type animals. The increased energy to failure was primarily caused by a large increase in post-yield behavior, with no difference in pre-yield behavior. The HBM mice had increased mineral-to-matrix and collagen cross-link ratios, and decreased crystallinity, carbonate, and acid phosphate substitution as measured by Fourier transform infrared microspectroscopy, but no differences in crystal length, intra-fibular strains, and mineral spacing compared to wild-type controls, as measured by X-ray scattering. The largest between genotype difference in material properties was a twofold increase in the modulus of toughness in HBM mice. Step-wise regression analyses showed that the specific matrix compositional parameters most closely associated with material properties varied between the wild-type and HBM genotypes. Although the mechanisms controlling the paradoxical combination of more mineralized yet tougher bone in HBM mice remain to be fully explained, the findings suggest that LRP5 represents a target to not only build bone mass but also to improve bone quality.