Michael R. Doschak

Incomplete penetrance and phenotypic variability characterize Gdf6-attributable oculo-skeletal phenotypes

Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.

In vivo microfocal computed tomography and micro–magnetic resonance imaging evaluation of antiresorptive and antiinflammatory drugs as preventive treatments of osteoarthritis in the rat

OBJECTIVE To determine whether treatment with an antiresorptive drug in combination with an antiinflammatory drug reduces periarticular bone and soft tissue adaptations associated with the progression of posttraumatic secondary osteoarthritis (OA). METHODS We used in vivo microfocal computed tomography (micro-CT) to map bony adaptations and in vivo micro-magnetic resonance imaging (micro-MRI) to examine joint inflammation in a rat model of surgically induced OA secondary to knee triad injury. We examined the arthroprotective effects of the bisphosphonates alendronate and risedronate and the nonsteroidal antiinflammatory drug (NSAID) meloxicam. RESULTS Micro-CT revealed reduced levels of periarticular trabecular bone loss in animals with knee triad injury treated with the bisphosphonate drugs alendronate or risedronate, or the NSAID meloxicam, compared with untreated animals. Alendronate treatment reduced bony osteophyte development. While risedronate as a monotherapy did not positively impact osteophytogenesis, combination therapy with risedronate and meloxicam reduced osteophyte severity somewhat. Micro-MRI revealed an increased, diffuse water signal in the epiphyses of untreated rats with knee triad injury 8 weeks after surgery, suggestive of a bone marrow lesion-like stimulus. In contrast, meloxicam-treated rats showed a significant reduction in fluid signal compared with both bisphosphonate-treated groups 8 weeks after surgery. Histologic analysis qualitatively confirmed the chondroprotective effect of both bisphosphonate treatments, showing fewer degradative changes compared with untreated rats with knee triad injury. CONCLUSION Our findings indicate that select combinations of bisphosphonate and NSAID drug therapy in the early stages of secondary OA preserve trabecular bone mass and reduce the impact of osteophytic bony adaptations and bone marrow lesion-like stimulus. Bisphosphonate and NSAID therapy may be an effective disease-modifying drug regimen if administered early after the initial injury.

Polyethylenimine-PEG coated albumin nanoparticles for BMP-2 delivery.

Bone Morphogenetic Protein-2 (BMP-2) plays an important role in stimulating new bone formation, and has been utilized in clinical bone repair by implantation. In this study, we report a nanoparticulate (NP) system for BMP-2 delivery based on bovine serum albumin (BSA) NPs stabilized with a poly(ethylene glycol) modified polyethylenimine (PEI-PEG) coating. PEI-PEG with different PEG substitutions were synthesized, and the cell viability assay showed PEG substitution greatly reduced the cytotoxicity of the native PEI. Furthermore, PEI-PEG coated BSA NPs demonstrated smaller size and decreased zeta potential compared to PEI-coated NPs. The bioactivity of the encapsulated BMP-2 and the toxicity of PEI-PEG coated NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using human C2C12 cells. The results indicated that BMP-2 remained bioactive in NPs and PEI-PEG coating was advantageous in reducing the NP toxicity as compared to PEI. A 7-day pharmacokinetics study showed the BMP-2 retention in PEI-PEG coated NPs was similar to the uncoated NPs, but lower than that of the PEI-coated NPs. The osteoinductivity of BMP-2 delivered in NPs was determined by subcutaneous implantation in rats, and the results revealed that PEI-PEG coated BSA NPs induced significant de novo bone formation after implantation, while PEI-coated NPs demonstrated much less bone formation. We conclude that BMP-2 delivered by PEGylated PEI-coated BSA NPs displays favorable biocompatibility and promotes new bone formation after implantation.

Pharmacokinetics and bone formation by BMP-2 entrapped in polyethylenimine-coated albumin nanoparticles

The osteoinductive growth factor, bone morphogenetic protein-2 (BMP-2), is capable of inducing de novo bone formation after implantation. A nanoparticulate (NP) system was developed for BMP-2 delivery based on NPs fabricated from bovine serum albumin (BSA) and stabilized by polyethylenimine (PEI) coating. In this study, the pharmacokinetics and osteoinductivity of BMP-2 delivered with different BSA NP formulations were determined by subcutaneous implantation in rats. A 7-day pharmacokinetics study showed that PEI coating on NPs effectively reduced the initial burst release of BMP-2 and prolonged the BMP-2 retention at implantation site. However, the uncoated BMP-2 NPs (BMP-2 loading of 1.44% w/w) were able to induce a robust ectopic bone formation, while no bone formation was found by the BMP-2 NPs coated with PEI. The toxicity of the PEI used for NP coating was determined to be the reason for lack of osteoinduction. Increasing BMP-2 loading (up to 5.76% w/w) was then employed to formulate NPs with lower PEI content; the higher BMP-2 loading was found to better promote induction of de novo bone. Our findings indicated that PEI coating on BSA NPs was effective for controlling BMP-2 release from NPs, but the toxicity of cationic PEI was a concern for the osteoinductive activity, which should be alleviated by further optimization of NP formulations.

Anabolic effects of low-intensity pulsed ultrasound on human gingival fibroblasts §

OBJECTIVE Low-intensity pulsed ultrasound (LIPUS) demonstrated anabolic effects on cementoblasts, odontoblasts, and periodontal ligament cells. However, LIPUS effect on human gingival fibroblasts (HGF) remains to be investigated. Therefore, we evaluated the in vitro effects of LIPUS on HGF proliferation and differentiation to test its feasibility for periodontal therapy. DESIGN LIPUS treatment (1.5MHz, 30mW/cm(2)) was applied to HGF in the experimental groups after 24-h of culture (5 or 10min/day for 28 days) and omitted in the control. Changes in HGF activities were evaluated in response to LIPUS treatment in dose-dependent (5 and 10min) and time-dependent (weeks 1-4) manner. The effects of LIPUS on HGF cell viability (MTT), proliferation (total DNA content and growth pattern), alkaline phosphatase (ALP) activity, and gene expression by reverse-transcriptase polymerase chain reaction (RT-PCR) were determined. RESULTS Cell viability remained unchanged after LIPUS treatment during the 4 weeks of treatment as compared to the untreated control group which ensured a safe biological response. Both LIPUS treatments (5-10min/day) did not yield any significant changes in the proliferation, and expression of proliferating cell nuclear antigen (PCNA) and collagen-I (COL-I). Conversely, LIPUS treatment enhanced osteogenic differentiation potential of HGF as determined by significant up-regulation of specific ALP activity and osteopontin (OPN) expression, with optimum effect following 3 weeks of 5min/day LIPUS treatment. CONCLUSION LIPUS treatment at 30mW/cm(2) selectively enhanced HGF differentiation but not proliferation. The ability of LIPUS to enhance HGF differentiation is promising for its application in cell-based periodontal therapy.

Synthesis, characterization and evaluation of bone targeting salmon calcitonin analogs in normal and osteoporotic rats.

In order to assess the therapeutic efficacy of an antiresorptive drug with imparted bone targeting potential using bisphosphonate (BP) conjugation and an improved pharmacokinetic profile using PEGylation, we synthesized, characterized and evaluated in vivo efficacy of bone-targeting PEGylated salmon calcitonin (sCT) analog (sCT-PEG-BP). sCT-PEG-BP was compared with non-PEGylated bone targeting sCT analog (sCT-BP) and unmodified, commercially available sCT. sCT-PEG-BP conjugates were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis. The effect of PEG-BP or BP upon sCT secondary structure was examined by Circular Dichroism and sCT-PEG-BP was evaluated for in vitro bone mineral Hydroxyapatite (HA) binding ability and calcium salts specificity using a binding assay for bone HA and several calcium salts. Anti-calcitonin antibody binding ability of these analogs was determined using enzyme-linked immunosorbent assay (ELISA), by reacting bone targeting sCT analogs with calcium phosphate coated Osteologic® plates and detecting the bound sCT using anti-sCT antibody. Potential cytotoxicity of these compounds was evaluated in monocytic RAW 264.7 cells, and sCT bioactivity was evaluated using an in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. Finally, in vivo efficacy of each compound was evaluated by determining the plasma levels of calcium after s.c. administration in normal rats, and in a rat model of Osteoporosis, secondary to ovariectomy (OVX). In vivo micro-computed tomography (micro-CT) was used to temporally map and quantify alterations in bone volume and bone mineral density (BMD) in the same animals at 1, 4, 8 and 12 weeks after OVX surgery. Sixteen 6 week old virgin female rats underwent OVX surgery followed by the daily s.c. injection of 2.5IU/kg/day sCT or equivalent analogs, and compared to four sham-operated, placebo treated control rats. Our results showed the chemical coupling of PEG-BP or BP to sCT altered its secondary structure without altering its antibody binding ability. sCT analogs retained strong sCT bioactivity, were non-toxic to RAW 264.7 cells in culture and elicited a comparable hypocalcemic effect to that of unmodified sCT in normal rats. Compared to marketed unmodified sCT, sCT-PEG-BP showed significantly improved efficacy in terms of preserving bone volume, BMD and trabecular micro-architecture in osteoporotic rats at the initial dose tested. Bisphosphonate-mediated targeting of PEGylated sCT to bone represents a new class of targeted antiresorptive compounds that has not previously been attempted.

Potential mechanism of alendronate inhibition of osteophyte formation in the rat model of post-traumatic osteoarthritis: evaluation of elemental strontium as a molecular tracer of bone formation

OBJECTIVE To employ elemental Strontium as a tracer of bone turnover, in the presence (or absence) of the bisphosphonate drug Alendronate, in order to spatially map osteophytogenesis and other bone turnover in rats developing post-traumatic secondary osteoarthritis (PTOA). METHODS PTOA was induced in rats by medial meniscectomy surgery. We utilized in-vivo microfocal computed tomography (CT) to follow bony adaptations in groups for 8 weeks after surgery, either with or without alendronate treatment. Electron probe microanalysis (EPMA) was used to detect Strontium incorporation in mineralizing tissues. Histologic studies were conducted on the same samples using Safranin-O/fast green and Tetrachrome staining of decalcified sections to examine articular cartilage health and osteophyte formation at the sites of elemental Strontium deposition. RESULTS EPMA revealed uniform incorporation of Strontium over actively remodeling trabecular surfaces in normal control rats. That pattern was significantly altered after meniscectomy surgery resulting in greater Strontium signal at the developing osteophyte margins. Alendronate treatment inhibited osteophyte development by 40% and 51% quantified by micro-CT volumetric measurements at 4 and 8 weeks after surgery, respectively. Osteophytes in the alendronate group were more cartilaginous in composition [i.e., lower bone mineral density (BMD)] compared to the untreated group. Histological analysis confirmed the osteophyte inhibitory effect of alendronate, and also verified reduced degeneration of the articular cartilage compared to untreated rats. CONCLUSION Our study confirmed that alendronate administration will reduce osteophyte formation in a rat model of post-traumatic osteoarthritis, partially through the inhibition of secondary remodeling of osteophytes. Our study is the first to employ elemental Strontium as a tracer of bone turnover in the pathogenesis of osteoarthritis and to assess the efficacy of bisphosphonate antiresorptive drug interventions on osteophytogenesis.

Effect of Low-Intensity Pulsed Ultrasound on Orthodontically Induced Root Resorption in Beagle Dogs

We investigated the effect of low-intensity pulsed ultrasound (LIPUS) on orthodontically induced inflammatory root resorption in vivo. Ten beagle dogs were treated with an orthodontic appliance to move the mandibular fourth premolars bodily. The orthodontic movement was carried out for 4 wk with a continuous force of 1 N/side; using a split-mouth model, LIPUS was applied daily for 20 min. Fourth premolar and surrounding periodontal tissue were evaluated with micro-computed tomography and hematoxylin and eosin and tartrate-resistant acid phosphatase staining. We calculated the number, volume and distribution of root resorption lacunae and their percentage relative to total root volume, orthodontic tooth movement and periodontal ligament space. There was no significant difference in orthodontic tooth movement between the two sides. LIPUS significantly reduced the number of orthodontically induced inflammatory root resorption initiation areas by 71%, reduced their total volume by 68% and reduced their volume relative to the affected root total volume by 70%. LIPUS induced the formation of a precementum layer, thicker cementum and reparative cellular cementum.

Synthesis, characterization and in vitro evaluation of a bone targeting delivery system for salmon Calcitonin

Synthetic salmon Calcitonin (sCT) is currently used to treat and manage conditions associated with low bone mass, and elicits its antiresorptive effect by acting upon Calcitonin receptors (CTRs) located on bone-resorbing osteoclast cells. However, CTRs are also widely distributed in many non-skeletal tissues (such as kidney, brain, and lung), and the competitive uptake of available sCT amongst such CTRs likely reduces sCT availability for bone resident osteoclast cells, particularly if the drug is administered systemically and not specifically targeted to bone. Hence, the objective of this study was to synthesize and characterize a bisphosphonate (BP)-mediated bone targeting delivery system for sCT and to determine whether the bioactivity of sCT was retained after BP conjugation. BP-sCT conjugates were synthesized by initially reacting sCT with sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) in dimethyl formamide in the presence of triethylamine (TEA) at room temperature. Thiolated (Thiol)-BP was then reacted with the sCT-sulfo-SMCC conjugates to generate sCT-BP conjugates, which were purified by dialysis and assayed using the micro-BCA protein assay. Non-BP containing control sCT-Cysteine conjugates were also synthesized using the same procedure. Reactions were monitored and characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF) analysis and Tris-Tricine SDS-PAGE. Conjugates were evaluated for in vitro bone mineral affinity using a hydroxyapatite binding test, for bone mineral specificity using different calcium salt binding affinity assays, and for continued sCT bioactivity after conjugation using an intracellular cAMP stimulation in human T47D breast cancer cells. Our results confirmed that BP-conjugated sCT exhibited significantly greater affinity and specificity for bone mineral over unmodified sCT, and that sCT-BP conjugates retained strong CT bioactivity after conjugation. Our conjugation strategy holds the promise of facilitating the delivery of sCT preferentially to skeletal bony tissues, thereby increasing its local concentration to bone surfaces. This peptide hormone-bisphosphonate drug system represents a new class of antiresorptive drug that has not previously been attempted, nor has a bone targeting formulation of sCT been reported.

In Vitro Osteogenic Induction Of Human Gingival Fibroblasts For Bone Regeneration

Background And Objective: Periodontitis is an inflammatory disease causing bone loss, and is a primary cause of tooth loss. Gingival fibroblasts are readily available with minimal donor site morbidity and may be ideal for tissue engineering efforts in regenerating lost alveolar bone. Dexamethasone (Dex) is commonly employed for in vitro osteogenic induction of a variety of cells, but its effect on human gingival fibroblasts (HGF) is still controversial. Therefore, the aim of our study was to investigate the osteogenic differentiation of HGF following Dex treatment. Methods: Cultured HGFs were exposed to osteogenic medium containing a wide range of Dex concentrations (0.01-10 µM). The osteogenic phenotype was assessed based on changes in alkaline phosphatase (ALP) activity, the mRNA expression of selected extracellular matrix proteins critical for mineralization and the extent of extracellular mineralization (Von Kossa staining and Ca-content). Results: All assays showed a consistent and maximal osteogenic effect of Dex on HGF at 0.1 and 0.5 µM (weeks 3 and 4), as evidenced by significant osteopontin and osteocalcin expression and mineralization. Longer cultures (week 4) also yielded positive osteogenic effect of Dex at 0.01 µM. Moreover, ALP activity was significantly stimulated at 0.1 and 0.5 µM Dex initially after one week, but ALP was subsequently reduced under Dex. Higher Dex concentrations caused down regulation of osteogenic effects observed at the optimal (0.1-0.5 µM) concentrations. Conclusion: Under appropriate osteogenic conditioning, Dex treated HGFs could be a potential source of cells for cell-based therapy for periodontal bone regeneration.