Six Weeks of Daily Abaloparatide Treatment Increased Vertebral and Femoral Bone Mineral Density, Microarchitecture and Strength in Ovariectomized Osteopenic Rats.

Abaloparatide is a novel, potent and selective activator of parathyroid hormone receptor 1 (PTHR1) under clinical development for the treatment of osteoporosis. We assessed the effect of 6 weeks of abaloparatide on bone mass, microarchitecture, quality and strength in ovariectomized (OVX) rats. After 8 weeks of post-surgical bone depletion (baseline), OVX rats (n = 20-21/group) received daily subcutaneous vehicle (OVX-Veh) or abaloparatide at 5 or 20 µg/kg. Sham-operated control rats (n = 24) received vehicle. Areal bone mineral density (aBMD) of the lumbar spine (L4), total femur and femur diaphysis was measured at baseline and after 6 weeks of treatment. Femur and vertebral bone architecture and mechanical properties were assessed at the end of the treatment phase. At baseline, OVX-Veh rats exhibited significantly lower aBMD relative to Sham controls. Treatment of OVX rats with abaloparatide at 5 or 20 µg/kg/day increased aBMD dose-dependently in the lumbar spine, total femur and femur diaphysis to levels exceeding OVX-Veh or Sham controls. The abaloparatide 5 and 20 µg/kg groups had improved trabecular microarchitecture relative to OVX vehicle, with trabecular BV/TV exceeding OVX-Veh control values by 57 and 78 % (respectively) at the lumbar spine, and by 145 and 270 % at the distal femur. Femur diaphyseal cortical volume and thickness were significantly greater in the abaloparatide 20 µg/kg group relative to OVX vehicle or Sham controls. Bone strength parameters of the femur diaphysis, femur neck and L4 vertebra were significantly improved in the OVX-ABL groups relative to OVX-Veh controls. Bone mass-strength relationships and estimated intrinsic strength properties suggested maintained or improved bone quality with abaloparatide. These data demonstrate skeletal restoration via abaloparatide treatment of osteopenic OVX rats, in association with improved trabecular microarchitecture, cortical geometry and bone strength at sites that have clinical relevance in patients with osteoporosis.

Binding Selectivity of Abaloparatide for PTH-Type-1-Receptor Conformations and Effects on Downstream Signaling.

The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1-34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1-34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1-34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R(0) and RG, and that efficient binding to R(0) results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R(0) PTHR1 conformation than does PTH (1-34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1-34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone.

Tissue engineering of bone: an ectopic rat model.

Tissue engineering is attempting to recreate the complexity of living tissues. In order to test a variety of scaffolds or cells that are constantly being developed, we describe here a model where tissue engineering of bone in a non-osseous environment at subcutaneous thoracic site of DA rats generates. In this model, cell - matix interactions can mimic the normal cascade of bone development into a well organized ossicle like structure including newly formed bone marrow, during 3-4 weeks. Histogenesis of cartilage, bone and bone marrow is closely related to changes in molecular expression of essential early transcriptional regulators of osteoblast differentiation. We tested different organic, anorganic and polymeric scaffolds and their interaction with mesenchymal stem cells present in fresh bone marrow. In another series of experiments we tested mesenchymal populations separated from cultures of calvaria and periosteum for their ability to form bone in the same rat model. It is concluded that this in vivo model is very potent in studying cell-scaffold interactions affecting the temporal and spatial tissue engineering of bone.

Commentary on: periodontally accelerated osteogenic orthodontics (PAOO) - a clinical dilemma.

It is apparent that tooth movement is enhanced by procedures that elevate the remodeling of alveolar bone, and of periodontal and gingival fibrous tissues. The periodontally accelerated osteogenic orthodontics (PAOO) also termed as Wilckodontics, involves full-thickness labial and lingual alveolar flaps accompanied with limited selective labial and lingual surgical scarring of cortical bone (corticotomy). Most of the authors suggest that the RAP is the major stimulus for alveolar bone remodeling, enabling the PAOO. However, we propose that detachment of the bulk of dentogingival and interdental fibers from coronal part of root surfaces by itself should suffice to stimulate alveolar bone resorption mainly on its PDL surfaces, leading to widening of the periodontal ligament space which largely attributes to accelerated osteogenic orthodontics. Moreover this limited fiberotomy also disrupts transiently the positional physical memory of dentition (PPMD), allowing accelerated tooth movement. During retention period, a new biological and physical connectivity is generated that could be termed as new positional memory of the dental arch.

Highly porous bioresorbable scaffolds with controlled release of bioactive agents for tissue-regeneration applications.

Highly porous poly(DL-lactic-co-glycolic acid) films with controlled release of horseradish peroxidase (HRP) as a model protein have been successfully developed and studied. These films, which are prepared by freeze-drying inverted emulsions, are designed for use in tissue-regeneration applications. The effects of the emulsion's formulation and host polymer's characteristics on the film's microstructure and HRP release profile over 4 weeks were investigated. A dual pore size population is characteristic for most films, with large 12-18 microm pores and small 1.5-7 microm pores, and porosity in the range of 76-92%. An increase in the polymer content and its initial molecular weight, organic/aqueous (O:A) phase ratio and lactic acid content, or a decrease in the HRP content, all resulted in a decreased burst effect and a more moderate release profile. A simultaneous change in two or three of these formulation parameters (compared to a reference formulation) resulted in a synergistic effect on the HRP release profile. A constant HRP release rate was achieved when a composite film was used. Human gingival fibroblast adhesion to the films indicated good biocompatibility. Appropriate selection of the emulsion's parameters can therefore yield highly porous films with the desired protein-release behavior which can serve as scaffolds for bioactive agents in tissue-regeneration applications.

Metronidazole-loaded bioabsorbable films as local antibacterial treatment of infected periodontal pockets.

BACKGROUND: Periodontal disease is infectious in nature and leads to an inflammatory response. It arises from the accumulation of subgingival bacterial plaque and leads to the loss of attachment, increased probing depth, and bone loss. It is one of the world's most prevalent chronic diseases. In this study we developed and studied metronidazole-loaded 50/50 poly(DL-lactide-co-glycolide) (PDLGA), 75/25 PDLGA, and poly(DL-lactic acid) (PDLLA) films. These films are designed to be inserted into the periodontal pocket and treat infections with controlled-release metronidazole for >or=1 month. METHODS: The structured films were prepared using the solution-casting technique. Concentrated solutions and high solvent-evaporation rates were used to get most of the drug located in the bulk, i.e., in whole film's volume. The effects of copolymer composition and drug content on the release profile, cell growth, and bacterial inhibition were investigated. RESULTS: The PDLLA and 75/25 PDLGA films generally exhibited a low- or medium-burst release followed by a moderate release at an approximately constant rate, whereas the 50/50 PDLGA films exhibited a biphasic release profile. The drug released from films loaded with 10% weight/weight metronidazole resulted in a significant decrease in bacterial viability within several days. When exposed to human gingival fibroblasts in cell culture conditions, these films maintained their normal fibroblastic features. CONCLUSIONS: This study enabled the understanding of metronidazole-release kinetics from bioabsorbable polymeric films. The developed systems demonstrated good biocompatibility and the ability to inhibit Bacteroides fragilis growth; therefore, they may be useful in the treatment of periodontal diseases.

Topical application of drugs influencing cytoskeleton and cell contractility affects alveolar bone loss in rats.

BACKGROUND: Several studies have shown that sectioning bundles of collagen fibers in the marginal gingiva during surgical procedures in animals is a distinct stimulus for alveolar bone resorption. Normally, gingival and periodontal fibroblasts, which reside on these collagen fibers, create physiological traction forces generated by the cytoskeleton. By splitting the fibers, traction forces are released, inducing changes in the cytoskeleton and cell shape. In this study, four drugs were selected, including cytochalasin D, EDTA, sodium orthovanadate, and H-7, all influencing the cytoskeleton-integrin-extracellular matrix (ECM) pathway, for their ability to reduce alveolar bone loss by local application. METHODS: The drugs were applied locally only once at the site of mucoperiosteal flap surgery in a rat model. Cytochalasin D (1 microl/microl), EDTA (0.24 mg/microl), sodium orthovanadate (0.02 mg/microl), and H-7 (0.10 microl/microl), each separately, were carried by a protective paste and placed immediately after elevating the flap. The analysis of alveolar bone loss was performed 3 weeks after surgery by scanning the microradiographic films of the mandible cross-sections. The percentages of cross sections with no, moderate, or severe bone loss in treated in comparison to non-treated rats are presented. RESULTS: EDTA, sodium orthovanadate, and H-7 were significantly effective in reducing alveolar bone loss. They were effective in reducing the amount of severe bone loss by 53%, 20%, and 58% while increasing the number of sections with no bone loss by 25%, 23%, and 35%, respectively. Cytochalasin D reduced alveolar bone loss insignificantly. CONCLUSION: EDTA, sodium orthovanadate, and H-7 are effective in reducing alveolar bone loss in rats following mucoperiosteum surgery.

Hydrophobicity as a design criterion for polymer scaffolds in bone tissue engineering.

Porous polymeric scaffolds play a key role in most tissue-engineering strategies. A series of non-degrading porous scaffolds was prepared, based on bulk-copolymerisation of 1-vinyl-2-pyrrolidinone (NVP) and n-butyl methacrylate (BMA), followed by a particulate-leaching step to generate porosity. Biocompatibility of these scaffolds was evaluated in vitro and in vivo. Furthermore, the scaffold materials were studied using the so-called demineralised bone matrix (DBM) as an evaluation system in vivo. The DBM, which is essentially a part of a rat femoral bone after processing with mineral acid, provides a suitable environment for ectopic bone formation, provided that the cavity of the DBM is filled with bone marrow prior to subcutaneous implantation in the thoracic region of rats. Various scaffold materials, differing with respect to composition and, hence, hydrophilicity, were introduced into the centre of DBMs. The ends were closed with rat bone marrow, and ectopic bone formation was monitored after 4, 6, and 8 weeks, both through X-ray microradiography and histology. The 50:50 scaffold particles were found to readily accommodate formation of bone tissue within their pores, whereas this was much less the case for the more hydrophilic 70:30 counterpart scaffolds. New healthy bone tissue was encountered inside the pores of the 50:50 scaffold material, not only at the periphery of the constructs but also in the center. Active osteoblast cells were found at the bone-biomaterial interfaces. These data indicate that the hydrophobicity of the biomaterial is, most likely, an important design criterion for polymeric scaffolds which should promote the healing of bone defects. Furthermore, it is argued that stable, non-degrading porous biomaterials, like those used in this study, provide an important tool to expand our comprehension of the role of biomaterials in scaffold-based tissue engineering approaches.

The influence of nacre surface and its modification on bone apposition: a bone development model in rats.

BACKGROUND: Bone graft substitutes are currently used individually or in various combinations in reconstructing bone defects. Nacre, marine mineralized structure, was recently proposed as a very biocompatible and osteoinductive material for use in periodontal and implant surgery. Our aim was to investigate the interaction between natural nacre and fresh bone marrow, during bone development, in an ectopic site of DA rats. Surface modifications of nacre were tested. METHODS: Demineralized bone matrix (DBM) cylinders (demineralized cortex of diaphysis) prepared from rat femurs were filled with fresh marrow, which was removed from other 2-month old DA rat femurs. Natural nacre particle or nacre which was treated with HCl, phosphate buffer saline (PBS), and Ca(OH)2 to modify its surface was placed into the DBM cylinders. The cylinders were implanted subcutaneously at the thoracic region of growing DA rats. After 4 weeks the cylinders were surgically removed, fixed in buffered formalin, and x-rayed. Scans of the microradiographs and histological evaluation of the DBM cylinders including bone developed at the interface of nacre and its surface modifications were compared to marrow controls. RESULTS: The results show that natural nacre is a poor conductive biomaterial in a bone developmental environment. Nacre surface treated with Ca(OH)2 and PBS was found to be most biocompatible. In this group, new bone was apposed directly on the nacre surface and the total amount of bone was highest in comparison to other treatment groups. CONCLUSIONS: This study does not support previous observations that nacre is osseoinductive. Our model system seems to be very sensitive and capable of testing interaction between surface modifications of biomaterials and fresh marrow in the process of new bone development.

The influence of alendronate on bone formation and resorption in a rat ectopic bone development model.

BACKGROUND: Most bone grafting techniques that include bone marrow, alloplastic materials, and extracellular bone matrix produce new bone mass, filling bone defects unpredictably. In most cases, the new bone undergoes resorption due to low local strains, resulting in significant bone loss. Recently, it was shown that alendronate and other bisphosphonates reduce bone loss when administered systemically or locally. The aim of this study was to investigate whether alendronate is effective on bone formation or bone resorption. METHODS: A total of 64 rats were divided into 2 main groups. In all the rats, fresh bone marrow removed from DA young rats was placed into demineralized rat femur cylinders (DBMC) and implanted into subcutaneous sites of host DA rats, to form new bone. Group A served as an alendronate treatment group, and group B served as a non-treated control. Group A received 100 microl of 1.5 mg/ml alendronate solution at 1, 2, and 3 weeks (group A1) and at 3, 4, and 5 weeks (group A2). At designated times, the rats were sacrificed, and the implanted DBMC was dissected out of the thorax and processed for histological and microradiography image analysis. RESULTS: Alendronate given at 1, 2, and 3 weeks (during the bone formation phase) did not increase the amount of bone or the visual bone density in comparison to the time-matched control, after 4 and 8 weeks. When alendronate was injected at 3, 4, and 5 weeks, the bone mass increased by 70% and by 166% after 6 and 10 weeks, respectively, in comparison to the untreated control. The visual bone density in group A2 was maintained at the level of 140 +/- 15 at 6 weeks and 152 +/- 15 at 10 weeks. The matched, non-treated control group B2 was significantly lower, 106 +/- 20 and 108 +/- 15, respectively. The histological sections showed that alendronate treatment at 3, 4, and 5 weeks maintained the normal appearance of the ossicle at 6 and 10 weeks in comparison to the osteopenic bone appearance in the matched controls. CONCLUSIONS: This study suggests that alendronate is effective in inhibiting bone loss, but ineffective during the bone formation phase. We suggest, therefore, that alendronate should be administered in procedures where bone resorption is expected.

Strain relaxation of fibroblasts in the marginal periodontium is the common trigger for alveolar bone resorption: a novel hypothesis.

In summary, the present commentary proposes a hypothesis that alveolar bone remodeling and bone loss in periodontitis, periodontal surgery, and in orthodontic tooth movement is triggered by a common "strain relaxation" signaling pathway of gingival and periodontal fibroblasts. The abrupt splitting, degradation, or relaxation of collagen fibers in the marginal periodontium produces a "strain relaxation" signal in the local fibroblasts which reside on these fibers, activating an ECM-integrin-cytoskeleton pathway. A cascade of cellular reactions which lead to osteoclastic bone resorption starting on the inner aspect (periodontal) of the alveolar bone then persists. A novel therapeutic approach is suggested here by using locally delivered drugs intervening in the cell contractile apparatus.