Application of a BMP2-binding heparan sulphate to promote periodontal regeneration.

Periodontitis is the most common inflammatory disease that leads to periodontal defects and tooth loss. Regeneration of alveolar bone and soft tissue in periodontal defects is highly desirable but remains challenging. A heparan sulphate variant (HS3) with enhanced affinity for bone morphogenetic protein-2 (BMP2) that, when combined with collagen or ceramic biomaterials, enhances bone tissue regeneration in the axial and cranial skeleton in several animal models was reported previously. In the current study, establishing the efficacy of a collagen/HS3 device for the regeneration of alveolar bone and the adjacent periodontal apparatus and related structures was sought. Collagen sponges loaded with phosphate-buffered saline, HS3, BMP2, or HS3 + BMP2 were implanted into surgically-created intra-bony periodontal defects in rat maxillae. At the 6 week end- point the maxillae were decalcified, and the extent of tissue regeneration determined by histomorphometrical analysis. The combination of collagen/HS3, collagen/BMP2 or collagen/HS3 + BMP2 resulted in a three to four-fold increase in bone regeneration and up to a 1.5 × improvement in functional ligament restoration compared to collagen alone. Moreover, the combination of collagen/HS3 + BMP2 improved the alveolar bone height and reduced the amount of epithelial growth in the apical direction. The implantation of a collagen/ HS3 combination device enhanced the regeneration of alveolar bone and associated periodontal tissues at amounts comparable to collagen in combination with the osteogenic factor BMP2. This study highlights the efficacy of a collagen/HS3 combination device for periodontal regeneration that warrants further development as a point-of-care treatment for periodontitis-related bone and soft tissue loss.

Autologous bone marrow clot as an alternative to autograft for bone defect healing.

OBJECTIVES: Long bone defects often require surgical intervention for functional restoration. The 'gold standard' treatment is autologous bone graft (ABG), usually from the patient's iliac crest. However, autograft is plagued by complications including limited supply, donor site morbidity, and the need for an additional surgery. Thus, alternative therapies are being actively investigated. Autologous bone marrow (BM) is considered as a candidate due to the presence of both endogenous reparative cells and growth factors. We aimed to compare the therapeutic potentials of autologous bone marrow aspirate (BMA) and ABG, which has not previously been done. METHODS: We compared the efficacy of coagulated autologous BMA and ABG for the repair of ulnar defects in New Zealand White rabbits. Segmental defects (14 mm) were filled with autologous clotted BM or morcellized autograft, and healing was assessed four and 12 weeks postoperatively. Harvested ulnas were subjected to radiological, micro-CT, histological, and mechanical analyses. RESULTS: Comparable results were obtained with autologous BMA clot and ABG, except for the quantification of new bone by micro-CT. Significantly more bone was found in the ABG-treated ulnar defects than in those treated with autologous BMA clot. This is possibly due to the remnants of necrotic autograft fragments that persisted within the healing defects at week 12 post-surgery. CONCLUSION: As similar treatment outcomes were achieved by the two strategies, the preferred treatment would be one that is associated with a lower risk of complications. Hence, these results demonstrate that coagulated BMA can be considered as an alternative autogenous therapy for long bone healing.Cite this article: Z. X. H. Lim, B. Rai, T. C. Tan, A. K. Ramruttun, J. H. Hui, V. Nurcombe, S. H. Teoh, S. M. Cool. Autologous bone marrow clot as an alternative to autograft for bone defect healing. Bone Joint Res 2019;8:107-117. DOI: 10.1302/2046-3758.83.BJR-2018-0096.R1.

Repair of segmental ulna defects using a ß-TCP implant in combination with a heparan sulfate glycosaminoglycan variant.

Given the wide spread clinical use of ceramic-based bone void fillers, we sought to determine the efficacy of an FDA-approved ß-tricalcium phosphate bone graft substitute (JAX™) in combination with a carboxymethyl cellulose (CMC) handling agent that included a particular heparan glycosaminoglycan (GAG) variant, herein referred to as HS3. Having recently demonstrated efficacy of a combination collagen/HS3 device, we further aimed to determine the support that HS3 could offer a handling agent used to administer a more tissue-relevant bone void filler. This study evaluated the JAX™-HS3 combination device in 1.5 cm critical-sized defects in the ulna bones of 27 male New Zealand White rabbits. Treatment groups consisted of JAX™ applied with CMC alone, or JAX™ with CMC containing either 30 µg or 100 µg of the HS3 GAG. Data based on radiographic, µCT, mechanical, and histological analyses at 4 and 8 weeks post-surgery, clearly demonstrate enhanced new bone formation in the JAX™-HS3 combination treated defects compared to treatment with JAX™ alone. The efficacy of such a combination advocates for inclusion of HS3 in handling agents used in the preparation of various bone void fillers being used in orthopaedic surgery. STATEMENT OF SIGNIFICANCE: Synthetic bone grafts and demineralized bone matrices are gaining prominence as alternatives to autologous and allogeneic bone grafts and are frequently administered in granular form, necessitating their combination with a handling agent. Typical handling agents include glycerol, gelatin, cellulose, hyaluronic acid and lecithin, formulated as hydrogels, which can be further enhanced by the addition of heparan sulfate (HS) glycosaminoglycans that augment the osteostimulatory properties of the graft. Here we assessed the efficacy of ß-TCP granules combined with a hydrogel consisting of carboxymethyl cellulose and the HS variant (HS3) previously shown to enhance osteogenic healing. The data advocates for HS3 to be included during the formulation of hydrogel-based carriers that support the various bone void fillers being used in orthopaedic surgery.

Affinity-selected heparan sulfate for bone repair.

Bone morphogenetic protein (BMP)-2 is a potent bone healing compound produced at sites of bone trauma. Here we present a therapeutic strategy to harness the activity of endogenously produced BMP-2 by delivery of an affinity-matched heparan sulfate (HS) glycos aminoglycan biomaterial that increases the bioavailability, bioactivity and half-life of this growth factor. We have developed a robust, cost effective, peptide-based affinity platform to isolate a unique BMP-2 binding HS variant from commercially available preparations of HS, so removing the manufacturing bottleneck for their translation into the clinic. This affinity-matched HS enhanced BMP-2-induced osteogenesis through improved BMP-2 kinetics and receptor modulation, prolonged pSMAD signaling and reduced interactions with its antagonist noggin. When co-delivered with a collagen implant, the HS was as potent as exogenous BMP-2 for the healing of critical-sized bone defects in rabbits. This affinity platform can be readily tuned to isolate HS variants targeted ata range of clinically-relevant growth and adhesive factors.

De-sulfation of MG-63 cell glycosaminoglycans delays in vitro osteogenesis, up-regulates cholesterol synthesis and disrupts cell cycle and the actin cytoskeleton.

Glycosaminoglycan (GAG) sugars are largely responsible for the bioactivity of the proteoglycan proteins they decorate, and are particularly important for mediating the processes of cell attachment and growth factor signaling. Here, we show that chlorate-induced de-sulfation of GAGs expressed by MG-63 osteosarcoma cells results in delayed cell proliferation when the cells are exposed to chlorate for short or medium periods, but a disrupted mineralization without altered cell proliferation in response to long-term chlorate exposure. Analysis of GAG-binding growth factor activity indicated that chlorate disrupted BMP2/noggin signaling, but not FGF2 activity. Microarray analyses, which were confirmed by subsequent cell-based assays, indicated that chlorate predominantly disrupted the cell cycle and actin cytoskeleton and upregulated cholesterol synthesis, without affecting cell migration or attachment. Furthermore, we observed that disruption of the functions of the proteoglycan syndecan-4 replicated phenotypes induced by chlorate, implicating a primary role for this proteoglycan in providing bioactivity for these cells. J. Cell. Physiol. 219: 572-583, 2009. (c) 2009 Wiley-Liss, Inc.

The stimulation of healing within a rat calvarial defect by mPCL-TCP/collagen scaffolds loaded with rhBMP-2.

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly epsilon-caprolactone/tricalcium phosphate (mPCL-TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL-TCP/collagen scaffolds with 5 microg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing micro-computed tomography (microCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL-TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non-BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by microCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, micro-compression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL-TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect

Glycosaminoglycan composition changes with MG-63 osteosarcoma osteogenesis in vitro and induces human mesenchymal stem cell aggregation.

Osteogenic differentiation is coordinated by the exposure of cells to temporal changes in a combination of growth factors and elements within the extracellular matrix (ECM). Many of the key proteins that drive these changes share the property of being dependent on ECM glycosaminoglycans (GAGs) for their activity. Here, we examined whether GAGs isolated from proliferating, differentiating and mineralizing MG-63 osteosarcoma cells differed in their physical properties, and thus in their capacities to coordinate the osteogenic cascade both in human MG-63 osteosarcoma cells and primary human mesenchymal stem cells (hMSCs). Our results show that the size distribution of GAGs, the expression of GAG-carrying proteoglycan cores and the expression of enzymes involved in their modification systematically change as MG-63 cells mature in culture. When dosed back onto cells exogenously in soluble form, GAGs regulated MG-63 survival and growth in a dose-dependent manner, but not differentiation in either cell type. In contrast, hMSCs aggregated into distinct colonies when grown on GAG-coated substrates, while MG-63 cells did not. Heparin-coated substrates improved hMSC viability without inducing aggregation. These results suggest a complex role for GAGs in coordinating the emergence of the osteoblast phenotype, and provide further evidence for the use of heparans in bone tissue repair applications.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite biomaterials for bone tissue regeneration: in vitro performance assessed by osteoblast proliferation, osteoclast adhesion and resorption, and macrophage proinflammatory response.

The efficacy of composite materials for bone tissue engineering is dependent on the materials' ability to support bone regeneration whilst inducing a minimal inflammatory response. In this study we examined the in vitro osteogenic and inflammatory properties of poly(3-hydroxybutyrate-co-3-valerate) (PHBV) with various calcium phosphate-reinforcing phases: nano-sized hydroxyapatite (HA); submicron-sized calcined hydroxyapatite (cHA); and submicron-sized beta-tricalcium phosphate (beta-TCP), using bioassays of cultured osteoblasts, osteoclasts, and macrophages. Our study showed that the addition of a nano-sized reinforcing phase to PHBV, whilst improving osteogenic properties, also reduces the proinflammatory response. Proinflammatory responses of RAW264.7/ELAM-eGFP macrophages to PHBV were shown to be markedly reduced by the introduction of a reinforcing phase, with HA/PHBV composites having the lowest inflammatory response. Osteoclasts, whilst able to attach to all the materials, failed to form functional actin rings or resorption pits on any of the materials under investigation. Cultures of osteoblasts (MC3T3-E1) readily attached and mineralised on all the materials, with HA/PHBV inducing the highest levels of mineralization. The improved biological performance of HA/PHBV composites when compared with cHA/PHBV and beta-TCP/PHBV composites is most likely a result of the nano-sized reinforcing phase of HA/PHBV and the greater surface presentation of mineral in these composites. Our results provide a new strategy for improving the suitability of PHBV-based materials for bone tissue regeneration.

Temporal expression of proteoglycans in the rat limb during bone healing.

Proteoglycans found in the bone extracellular matrix and on the cell surface can complex with HBGFs such as the FGFs, TGFs and BMPs which are known to play key roles in regulating fracture healing. Here we have studied the expression of key PGs during the bone repair process in order to determine the relationship between PG expression and healing status. We created non-critical sized trephine defects just proximal to the distal end of the tibial crest of adult male Wistar rats and examined the healing process histologically as well as by monitoring the temporal expression of mRNA transcripts for ALP, OP and OC, together with HSPG, CSPG and FGF-FGF receptor expression. Following surgery, animals were allowed to recover, and then euthanized after 7, 14, 21 and 28 days post-surgery, at which time tissue was harvested for histological examination and total RNA extracted and the mRNA transcripts examined by quantitative real-time PCR. HS and CSPG expression was generally observed to increase in the days immediately following injury, reaching peak expression two weeks post-surgery. This was followed by a gradual return to basal levels by day 28. The expression patterns of PGs were broadly similar with those of ALP, OP and FGFRs. The increase of mRNA expression for many key PGs detected during bone healing coincided with the elevation of bone markers and FGFRs, and provides further evidence that PGs involved in bone repair act in part through susceptible growth factors, including the FGF/FGFR system. The data presented here indicates that increased proteoglycan expression is involved in the early stages of bone healing at a time when previous studies have shown that the levels of HBGFs are maximal. Hence there exists a rationale for an exploration of the use of exogenous PGs as an adjunct therapy to potentiate the powerful effects of these factors and to augment the natural healing response.

Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) supports in vitro osteogenesis.

Studies have demonstrated that polymeric biomaterials have the potential to support osteoblast growth and development for bone tissue repair. Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV), a bioabsorbable, biocompatible polyhydroxy acid polymer, is an excellent candidate that, as yet, has not been extensively investigated for this purpose. As such, we examined the attachment characteristics, self-renewal capacity, and osteogenic potential of osteoblast-like cells (MC3T3-E1 S14) when cultured on PHBV films compared with tissue culture polystyrene (TCP). Cells were assayed over 2 weeks and examined for changes in morphology, attachment, number and proliferation status, alkaline phosphatase (ALP) activity, calcium accumulation, nodule formation, and the expression of osteogenic genes. We found that these spindle-shaped MC3T3-E1 S14 cells made cell-cell and cell-substrate contact. Time-dependent cell attachment was shown to be accelerated on PHBV compared with collagen and laminin, but delayed compared with TCP and fibronectin. Cell number and the expression of ALP, osteopontin, and pro-collagen alpha1(I) mRNA were comparable for cells grown on PHBV and TCP, with all these markers increasing over time. This demonstrates the ability of PHBV to support osteoblast cell function. However, a lag was observed for cells on PHBV in comparison with those on TCP for proliferation, ALP activity, and cbfa-1 mRNA expression. In addition, we observed a reduction in total calcium accumulation, nodule formation, and osteocalcin mRNA expression. It is possible that this cellular response is a consequence of the contrasting surface properties of PHBV and TCP. The PHBV substrate used was rougher and more hydrophobic than TCP. Although further substrate analysis is required, we conclude that this polymer is a suitable candidate for the continued development as a biomaterial for bone tissue engineering.

Comparisons between bone and cementum compositions and the possible basis for their layered appearances.

In humans, age estimation from the adult skeleton represents an attempt to determine chronological age based on growth and maturational events. In teeth, such events can be characterized by appositional growth layers in midroot cementum. The purpose of this study was to determine the underlying cause of the layered microstructure of human midroot cementum. Whether cementum growth layers are caused by changes in relative mineralization, collagen packing and/or orientation, or by variations in organic matrix apposition was investigated by subjecting midroot sections of human canine teeth to analysis using polarized light and scanning electron microscopy (SEM). Polarized light was used to examine transverse midroot sections in both mineralized and demineralized states. Mineralized sections were also reexamined following subsequent decollagenization. Polarized light was additionally used in the examination of mineralized sections taken transversely, longitudinally, and obliquely from the same tooth root. From the birefringence patterns it was concluded that collagen orientation does not change with varying section plane. Instead, the mineral phase was most responsible for the birefringence of the cementum. SEM studies suggested that neither collagen packing nor collagen orientation change across the width of the cementum, confirming and validating the results of the polarized light examination. Also, SEM analysis using electron backscatter and the electron probe suggested no changes in the mean atomic number density, calcium, phosphate, and sulfur levels across the width of the cementum. Therefore, we conclude that crystalline orientation and/or size is responsible for the layered appearance of cementum.

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways.

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-kappaB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

The proliferative and migratory activities of breast cancer cells can be differentially regulated by heparan sulfates.

To explore how heparan sulfate (HS) controls the responsiveness of the breast cancer cell lines MCF-7 and MDA-MB-231 to fibroblast growth factors (FGFs), we have exposed them to HS preparations known to have specificity for FGF-1 (HS glycosaminoglycan (HSGAG A)) or FGF-2 (HSGAGB). Proliferation assays confirmed that MCF-7 cells were highly responsive to FGF-2 complexed with GAGB, whereas migration assays indicated that FGF-1/HSGAGA combinations were stimulatory for the highly invasive MDA-MB-231 cells. Quantitative polymerase chain reaction for the levels of FGF receptor (FGFR) isoforms revealed that MCF-7 cells have greater levels of FGFR1 and that MDA-MB-231 cells have greater relative levels of FGFR2. Cross-linking demonstrated that FGF-2/HSGAGB primarily activated FGFR1, which in turn up-regulated the activity of mitogen-activated protein kinase; in contrast, FGF-1/HSGAGA led to the phosphorylation of equal proportions of both FGFR1 and FGFR2, which in turn led to the up-regulation of Src and p125(FAK). MDA-MB-231 cells were particularly responsive to vitronectin substrates in the presence of FGF-1/HSGAGA, and blocking antibodies established that they used the alpha(v)beta(3) integrin to bind to it. These results suggest that the clustering of particular FGFR configurations on breast cancer cells induced by different HS chains leads to distinct phenotypic behaviors.

Microscopic age changes in the human occipital bone.

The value of histological examination of the human occipital bone for estimation of age-at-death was assessed. Undecalcified sections of occipital bone from eighteen male Caucasian subjects between the ages of 21 and 70 years were prepared for analysis using polarized light microscopy. The fractional volumes of primary osteons, secondary osteons, osteon fragments, and lamellar bone in both the outer and inner cortical tables were determined. It was found that with increasing age there is a decrease in the fractional volume of primary osteons and a significant decrease in the fractional volume of lamellar bone. The fractional volume of secondary osteons was not found to change significantly with age, while the fractional volume of osteon fragments significantly increases. The microscopic results reflect the continuous process of bone remodeling that is responsible for the variation in cortical parameters with age and is the primary basis for age predicting methods. While observable changes in the occipital bone do occur with increasing age, the amount of random variation in the parameters examined preclude their use for accurate age estimation.