Advanced Bone Formation Research Articles

Osteogenic Effect of a Bioactive Calcium Alkali Phosphate Bone Substitute in Humans.

(1) Background: The desire to avoid autograft harvesting in implant dentistry has prompted an ever-increasing quest for bioceramic bone substitutes, which stimulate osteogenesis while resorbing in a timely fashion. Consequently, a highly bioactive silicon containing calcium alkali orthophosphate (Si-CAP) material was created, which previously was shown to induce greater bone cell maturation and bone neo-formation than β-tricalcium phosphate (β-TCP) in vivo as well as in vitro. Our study tested the hypothesis that the enhanced effect on bone cell function in vitro and in sheep in vivo would lead to more copious bone neoformation in patients following sinus floor augmentation (SFA) employing Si-CAP when compared to β-TCP. (2) Methods: The effects of Si-CAP on osteogenesis and Si-CAP resorbability were evaluated in biopsies harvested from 38 patients six months after SFA in comparison to β-TCP employing undecalcified histology, histomorphometry, and immunohistochemical analysis of osteogenic marker expression. (3) Results: Si-CAP as well as β-TCP supported matrix mineralization and bone formation. Apically furthest away from the original bone tissue, Si-CAP induced significantly higher bone formation, bone-bonding (bone-bioceramic contact), and granule resorption than β-TCP. This was in conjunction with a higher expression of osteogenic markers. (4) Conclusions: Si-CAP induced higher and more advanced bone formation and resorbability than β-TCP, while β-TCP's remarkable osteoconductivity has been widely demonstrated. Hence, Si-CAP constitutes a well-suited bioactive graft choice for SFA in the clinical arena.

Crosstalk between microRNA-21–5p and the transcription factor Dec1 maintains osteoblast function

MicroRNAs are associated with pivotal post-transcriptional gene regulation in bone formation. Human differentiated embryonic chondrocyte expressed gene 1 (Dec1) is also involved in regulating osteoblastogenesis. In the present study, we aimed to investigate the distinctive role of miR-21–5p and Dec1 in osteoblast function and to determine their biological functions. MC3T3-E1 pre-osteoblastic cells were used for in vitro analyses. miR-21–5p knockout (KO) mice, Dec1KO mice and age-matched wild-type (WT) mice were used to characterize the influence of miR-21–5p and Dec1 deficiencies on bone formation. Morphological analyses [micro-computed tomography (micro-CT)] were performed, and measurements were collected to validate miR-21-5pKO mice. Histopathological changes in mouse femur tissues were assessed by H-E staining, Azan staining, Masson's Trichrome staining, and Toluidine Blue staining. Quantitative real-time RT-PCR, western blotting and immunohistochemical staining were used to characterize the expression levels of Alkaline Phosphatase, Runx2, Osterix, Osteopontin, Dec1 and miR-21–5p. Bioinformatics analyses and dual-luciferase reporter assays were performed to confirm Dec1 as a target of miR-21–5p. Dec1 expression was gradually increased from day 7 of osteoblast induction, while miR-21–5p showed a peak at day 21. In non-induced osteoblasts, a mechanistically gain-of-function transfection study with a miR-21–5p mimic enhanced Runx2 and Osterix expression but suppressed Dec1. miR-21-5pKO mice had reduced bone growth. Dec1-deficient mice showed advanced bone formation at the age of 12 weeks compared to WT mice. The Dec1 deficiency upregulated Runx2 and Osterix expression in Dec1KO mouse femurs. Those changes, however, were reversed in miR-21-5pKO mouse femurs compared to WT mouse femurs. Dual-luciferase reporter assays showed that Dec1 is a possible downstream target of miR-21–5p. These findings showed that the reduced osteogenic potential due to a miR-21–5p deficiency is achieved by enhanced Dec1 expression and that the miR-21–5p/Dec1 axis is involved in regulating osteoblast function.

Cellular and Molecular Dynamics during Early Oral Osseointegration: A Comprehensive Characterization in the Lewis Rat.

There is a need to improve the predictability of osseointegration in implant dentistry. Current literature uses a variety of in vivo titanium (Ti) implantation models to investigate failure modes and test new materials and surfaces. However, these models produce a variety of results, making comparison across studies difficult. The purpose of this study is to validate an oral osseointegration in the Lewis rat to provide a reproducible baseline to track the inflammatory response and healing of Ti implants. Ti screws (0.76 mm Ø × 2 mm length) were implanted into the maxillary diastema of 52 adult male Lewis rats. Peri-implant tissues were evaluated 2, 7, 14, and 30 days after implantation (n = 13). Seven of the 13 samples underwent microtomographic analysis, histology, histomorphometry, and immunohistochemistry to track healing parameters. The remaining six samples underwent quantitative polymerase chain reaction (qPCR) to evaluate gene expression of inflammation and bone remodeling markers over time. This model achieved a 78.5% success rate. Successful implants had a bone to implant contact (BIC)% of 68.86 ± 3.15 at 30 days on average. Histologically, healing was similar to other rodent models: hematoma and acute inflammation at 2 days, initial bone formation at 7, advanced bone formation and remodeling at 14, and bone maturation at 30. qPCR indicated the highest expression of bone remodeling and inflammatory markers 2-7 days, before slowly declining to nonsurgery control levels at 14-30 days. This model combines cost-effectiveness and simplicity of a rodent model, while maximizing BIC, making it an excellent candidate for evaluation of new surfaces.

EFFECT OF A BIOACTIVE CALCIUM ALKALI ORTHOPHOSPHATE BONE GRAFTING MATERIAL AS COMPARED TO TRICALCIUM PHOSPHATE ON OSTEOGENESIS AFTER SINUS FLOOR AUGMENTATION IN PATIENTS

Sinus floor augmentation (SFA) has become a well-recognized procedure for site development in the atrophic posterior maxilla prior to dental implant placement. There has been an increasing search for relatively rapidly resorbable bone substitutes that enhance bone formation and facilitate bone regeneration with complete replacement by functional bone tissue. This has led to the development of a glassy crystalline silica-containing calcium alkali orthophosphate (Si-CAOP). The specific aim of this study was to assess histologically the effect of this grafting material on osteogenesis and osteogenic marker expression and to compare this effect to that of β-tricalcium phosphate (β-TCP) in human biopsies obtained 6 months after SFA. Cylindrical biopsies, harvested 6 months after SFA, were processed for immunohistochemical analysis of sawed hard tissue sections using primary antibodies specific to osteocalcin (OC), collagen type I (Col I), bone sialoprotein (BSP), and alkaline phosphatase (ALP). Furthermore, the bone and particle area fraction were measured histomorphometrically in all biopsies in order to characterize bone formation and degradation of the bone grafting materials. Both biomaterials facilitated osteoblast differentiation and bone regeneration of resorbed alveolar ridges, resulting in sufficient bone formation for supporting dental implants. Si-CAOP specimens displayed more advanced bone formation and significantly greater particle degradation than TCP sites. This was accompanied by significantly higher expression of OC, Col I, BSP, and ALP in the osteoid in contact with the degrading particles. These promising results indicate that the Si-CAOP material has great potential use for SFA in humans, thereby confirming its superiority to β-TCP. A prospective study involving a larger patient number, a split-mouth design and analysis of angiogenic properties and of cone-beam CT-data for assessing the volume stability of the augmented area is warranted to further confirm the high osteogenic capacity of Si-CAOP for orofacial bone regeneration.

The effects of tubular structure on biomaterial aided bone regeneration in distraction osteogenesis

Long duration of bone consolidation in distraction osteogenesis (DO) has been regarded as a major disadvantage. Porous bone substitutes have been long used in aiding bone formation. However the effects of geometric tubular structure of the porous scaffolds on bone formation in DO remains unknown. This study investigated the effects of porous scaffolds with three different geometric structures on bone regeneration in a rabbit DO model. β-tricalcium phosphate (β-TCP) was fabricated into no tubular, single tubular, or multi-tubular structure constructs. The microstructure of the three types of β-TCP constructs was determined by scanning electron microscope (SEM). Unilateral tibial osteotomy was made at the midshaft with 1 cm tibial shaft removed, fixed with an external lengthener in adulkt New Zealand white rabbits. After acute tibial shortening of 0.5 cm, the remaining 0.5 cm defect was implanted with β-TCP constructs with no tubular, single tubular, or multi-tubular structure. Five days after surgery, the tibia was lengthened at 0.5 mm/12 h for 5 days. After further 28 days of consolidation, the animals were terminated. Bilateral tibial specimens were collected for X-ray, micro-computed tomography examination, four-point bending mechanical test and histological analysis. SEM results showed that pore sizes and pore interconnectivity in all three types of β-TCP constructs were identical. 38 days after surgery (4 weeks after lengthening), the new bone formation and mechanical properties were significantly enhanced in the groups implanted with multi-tubular and single tubular constructs, comparing to the group implanted with no tubular constructs. Furthermore, the group implanted with multi-tubular constructs had significantly better mechanical properties comparing to the group implanted with single tubular constructs. Histological results confirmed that group implanted with multi-tubular constructs had the most advanced bone formation and remodelling in the defect gap. The current study confirmed that β-TCP constructs with multi-tubular structure was the best choice as scaffold aid for bone regeneration in DO, indicating a novel fabrication approach of multi-tubular constructs for rapid bone formation together with DO procedure. Porous β-TCP scaffold with a multi-tubular structure used in combination with DO procedure can significantly shorten the bone consolidation duration. The tubular shaped porous β-TCP scaffold may be used to fill bone defect and to aid bone formation, consolidaiton and remodelling in DO.

Recombinant Human BMP6 Applied Within Autologous Blood Coagulum Accelerates Bone Healing: Randomized Controlled Trial in High Tibial Osteotomy Patients.

ABSTRACTBone morphogenetic proteins (BMPs) are potent osteogenic proteins that induce new bone formation in vivo. However, their effect on bone healing in the trabecular bone surfaces remains challenging. We evaluated the safety and efficacy of recombinant human BMP6 (rhBMP6) applied within an autologous blood coagulum (ABC) in a surgically created wedge defect of the proximal tibia in patients undergoing high tibial osteotomy (HTO) for varus deformity and medial osteoarthritis of the knee. We enrolled 20 HTO patients in a randomized, placebo‐controlled, double‐blinded phase I/II clinical trial. RhBMP6/ABC (1.0 mg/10 mL ABC prepared from peripheral blood) or placebo (10 mL ABC containing excipients) was administered into the tibial wedge defects. Patients were followed for 0 to 24 months by clinical examination (safety) and computed tomography (CT) and serial radiographic analyses (efficacy). The results show that there were no detectable anti‐rhBMP6 antibodies in the blood of any of the 20 patients at 14 weeks after implantation. During the 24 months of follow‐up, there were no serious adverse reactions recorded. The CT scans from defects of patients treated with rhBMP6/ABC showed an accelerated bone healing compared with placebo at 9 weeks (47.8 ± 24.1 versus 22.2 ± 12.3 mg/cm3; p = 0.008) and at 14 weeks (89.7 ± 29.1 versus 53.6 ± 21.9 mg/cm3; p = 0.006) follow‐up. Radiographic analyses at weeks 6 and 24 and months 12 and 24 suggested the advanced bone formation and remodeling in rhBMP6/ABC‐treated patients. In conclusion, we show that rhBMP6/ABC at a dose of 100 μg/mL accelerated bone healing in patients undergoing HTO without serious adverse events and with a good tolerability compared with placebo alone. Overall, for the first time, a BMP‐based osteogenic implant was examined against a placebo for bone healing efficacy in the trabecular bone surface, using an objective bone mineral density measurement system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

The influence of locally applied granulocyte-colony stimulating factoron osteoporotic bone.

The compromised capacity of bone healing in osteoporotic population renders a serious concern of patients and clinicians. This study aimed to investigate the influence of G-CSF on bone reconstruction using an osteoporotic animal model. Sixty skeletal mature female Spraque-Dawley rats underwent bilateral ovariectomy (OVX) and were assigned into three groups (n=20). Three months after OVX, defects of 5mm in cranial and 2mm in femur were surgically created on all the animals. The defects were left unfilled, filled with gelatin sponge (GS), or filled with granulocyte-colony stimulating factor (G-CSF) infused GS. Specimens were retrieved for histomorphometric and micro-CT analyses at weeks 1, 4, 8, and 12 after surgery. At early stage of week 1 to week 8, the histomorphometric and micro-CT analysis demonstrated more advanced bone formation in femur in the control group; by week 12, all groups achieved cortical closure. In cranial bone, more advanced bone formation was exhibited in G-CSF-treated group at both early and late stages, although this observation was not statistically significant. The results indicated that in osteoporotic bone, G-CSF may advance bone healing in cranial bone where spontaneous bone formation was insufficient.

Cell-based therapy by implanted human bone marrow-derived mononuclear cells improved bone healing of large bone defects in rats.

QUESTION/AIM: Cell-based therapy by cultivated stem cells (mesenchymal stem cells [MSC] and endothelial progenitor cells [EPC]) in a large-sized bone defect has already shown improved vascularization and new bone formation. However, these methods are clinically afflicted with disadvantages. Another heterogeneous bone marrow cell population, the so-called human bone marrow-derived mononuclear cells (BMC), has nevertheless been used clinically and showed improved vascularization in ischemic limbs or in the myocardium. For clinical use, a certified process has been established; thus, BMC were isolated from bone marrow aspirate by density gradient centrifugation, washed, cleaned, and given back to patients within several hours. This investigation tested the ability of human BMC seeded on beta-tricalcium phosphate (β-TCP) and placed into a large bone defect in rats to improve the bone healing process in vivo. Human EPC were isolated from buffy coat, and MSC or BMC, respectively, were isolated from bone marrow aspirate by density gradient centrifugation. 1.0×10(6) cells were loaded onto 750 μL β-TCP (0.7-1.4 mm). Large femoral defects (6 mm) in athymic rats were created surgically and stabilized with an internal fixateur. The remaining defects were filled with β-TCP granules alone (group 1), β-TCP+EPC/MSC (group 2), or β-TCP+BMC (group 3). After 8 weeks, histomorphometric analysis (new bone formation), radiological microcomputer tomography analysis (bony bridging), and biomechanical testing (three-point bending) were achieved. Moreover, a tumorigenicity study was performed to evaluate the safety of BMC implantation after 26 weeks. For statistical analysis, the Kruskal-Wallis test was used. Eight weeks after implantation of EPC/MSC or BMC, respectively, we detected a more significant new bone formation compared to control. In group 2 and 3, bony bridging of the defect was seen. In the control group, more chondrocytes and osteoid were detected. In the BMC and EPC/MSC group, respectively, less chondrocytes and a significantly more advanced bone formation were observed. The biomechanical stability of the bone regenerate was significantly enhanced if BMC and EPC/MSC, respectively, were implanted compared to control. Moreover, no tumor formation was detected either macroscopically or histologically after 26 weeks of BMC implantation. Implanted BMC suggest that a heterogeneous cell population may provide a powerful cellular therapeutic strategy for bone healing in a large bone defect in humans.

Osteoconductivity and Biodegradability of Collagen Scaffold Coated with Nano‐β‐TCP and Fibroblast Growth Factor 2

Nanoparticle bioceramics have become anticipated for biomedical applications. Highly bioactive and biodegradable scaffolds would be developed using nanoparticles of β‐tricalcium phosphate (β‐TCP). We prepared collagen scaffolds coated by nano‐β‐TCP and fibroblast growth factor 2 (FGF2) and evaluated the effects on new bone augmentation and biodegradation. The collagen sponge was coated with the nano‐TCP dispersion and freeze‐dried. Scaffold was characterized by SEM, TEM, XRD, compressive testing and cell seeding. Subsequently, the nano‐β‐TCP/collagen scaffold, collagen sponge, and each material loaded with FGF2 were implanted on rat cranial bone. As a control, no implantation was performed. Nano‐TCP particles were found to be attached to the fibers of the collagen sponge by SEM and TEM observations. Scaffold coated with nano‐TCP showed higher compressive strength and cytocompatibility. In histological evaluations at 10 days, inflammatory cells were rarely seen around the residual scaffold, suggesting that the nano‐TCP material possesses good tissue compatibility. At 35 days, bone augmentation and scaffold degradation in histological samples receiving nano‐β‐TCP scaffold were significantly greater than those in the control. By loading of FGF2, advanced bone formation is facilitated, indicating that a combination with FGF2 would be effective for bone tissue engineering.

Advanced Bone Formation in Mice with a Dominant-negative Mutation in the Thyroid Hormone Receptor β Gene due to Activation of Wnt/β-Catenin Protein Signaling

Thyroid hormone (T(3)) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance, but the signaling pathways mediating these effects are poorly understood. Thrb(PV/PV) mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T(3) receptor (TRβ(PV)) that cannot bind T(3) and interferes with the actions of wild-type TR. Thrb(PV/PV) mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and Thrb(PV/PV) mice. Activation of the canonical Wnt signaling in Thrb(PV/PV) mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during postnatal growth. By contrast, T(3) treatment inhibited Wnt signaling in osteoblastic cells, suggesting that T(3) inhibits the Wnt pathway by facilitating proteasomal degradation of β-catenin and preventing its accumulation in the nucleus. Activation of the Wnt pathway in Thrb(PV/PV) mice, however, results from a gain of function for TRβ(PV) that stabilizes β-catenin despite the presence of increased thyroid hormone levels. These studies demonstrate novel interactions between T(3) and Wnt signaling pathways in the regulation of skeletal development and bone formation.

RhBMP-2 not alendronate combined with HA-TCP biomaterial and distraction osteogenesis enhance bone formation

The long treatment duration of distraction osteogenesis (DO) usually causes some complications such as re-fracture, non-union. We have previously demonstrated that the combined use of biomaterial with distraction osteogenesis technique can enhance bone formation and consolidation. This study further tested whether the use of biological agents such as rhBMP-2 or alendronate together with biomaterials in DO will further promote bone formation. A 1.0-cm tibial shaft was removed in the left tibia of 30 rabbits. The 1.0-cm defect gap was reduced to 0.5 cm and the remaining 0.5-cm defect gap was filled with 0.5-cm restorable hydroxyapatite/tri-calcium phosphates (HA/TCP) cylindrical block. The animals were divided into three groups with the following added on the HA/TCP block: Group A 50 μl of saline, Group B 75 μg rhBMP-2 in 50 μl of saline, Group C 250 μg alendronate in 50 μl saline. The tibia was then fixed with unilateral lengthener and lengthening started 7 days after at a rate of 1.0 mm/day for 5 days. All animals were terminated at day 37 following surgery. The excised bone specimens were subject to micro-CT, mechanical testing and histological examinations. Bone mineral density and content were significantly higher in Groups A and B compared to Group C and the mechanical properties of the regenerates in Group B were highest. Micro-CT and histological examinations also confirmed that the regenerates in Group B had the most advanced bone formation, consolidation and remodeling comparing to other groups. The combined use of rhBMP-2 with HA-TCP biomaterial in DO has significantly enhanced bone formation and consolidation than using the HA-TCP biomaterials alone, whereas the use of alendronate has inhibitory effects on bone formation.

Novel application of HA‐TCP biomaterials in distraction osteogenesis shortened the lengthening time and promoted bone consolidation

This study tested the hypothesis that use of biomaterials in distraction osteogenesis (DO) would reduce the treatment time and enhance bone formation quality. A 1.0-cm tibial shaft was removed in the left tibia of 36 rabbits. Rabbits were randomly divided into three groups: group A, the defect gap was reduced with the tibia shortened for 1.0-cm; group B, the defect gap was filled with 1.0-cm restorable porous hydroxyapatite and Tri-calcium phosphates cylindrical block (HA/TCP block, diameter is 0.5-cm); group C, The 1.0-cm defect gap was reduced 0.5 cm and the remaining 0.5-cm defect gap was filled with the 0.5-cm HA/TCP block. The tibia was then fixed with unilateral lengthener; for groups A and C; lengthening started 7 days after surgery at a rate of 1.0 mm/day, in two steps. Group A received lengthening for 10 days and group C for 5 days, there was no lengthening for group B. All animals were terminated at day 37 following surgery. The excised bone specimens were subject to microcomputed tomography (micro-CT), mechanical testing, and histological examinations. Bone mineral density and content and tissue mineral density and content, as well as the mechanical properties of the regenerates were significantly higher in group C compared to groups A and B. Micro-CT and histological examinations also confirmed that the regenerates in Group C had most advanced bone formation, consolidation, and remodeling compared to other groups. In conclusion, the combined use of biomaterials and DO technique can reduce the treatment time and enhance bone consolidation in bone defect management.

Novel application of HA-TCP biomaterials in distraction osteogenesis shortened the lengthening time and promoted bone consolidation

This study tested the hypothesis that use of biomaterials in distraction osteogenesis (DO) would reduce the treatment time and enhance bone formation quality. A 1.0-cm tibial shaft was removed in the left tibia of 36 rabbits. Rabbits were randomly divided into three groups: group A, the defect gap was reduced with the tibia shortened for 1.0-cm; group B, the defect gap was filled with 1.0-cm restorable porous hydroxyapatite and Tri-calcium phosphates cylindrical block (HA/TCP block, diameter is 0.5-cm); group C, The 1.0-cm defect gap was reduced 0.5 cm and the remaining 0.5-cm defect gap was filled with the 0.5-cm HA/TCP block. The tibia was then fixed with unilateral lengthener; for groups A and C; lengthening started 7 days after surgery at a rate of 1.0 mm/day, in two steps. Group A received lengthening for 10 days and group C for 5 days, there was no lengthening for group B. All animals were terminated at day 37 following surgery. The excised bone specimens were subject to microcomputed tomography (micro-CT), mechanical testing, and histological examinations. Bone mineral density and content and tissue mineral density and content, as well as the mechanical properties of the regenerates were significantly higher in group C compared to groups A and B. Micro-CT and histological examinations also confirmed that the regenerates in Group C had most advanced bone formation, consolidation, and remodeling compared to other groups. In conclusion, the combined use of biomaterials and DO technique can reduce the treatment time and enhance bone consolidation in bone defect management.

Local injection of thrombin‐related peptide (TP508) in PPF/PLGA microparticles–enhanced bone formation during distraction osteogenesis

We have previously demonstrated that injections of the thrombin-related peptide, TP508, into the lengthening gap have significantly enhanced bone consolidation in a rabbit model of distraction osteogenesis. This study was to further test the effect of a single TP508 injection in slow release preparation on bone formation during distraction osteogenesis. Rabbits had left tibiae lengthened unilateral lengthener at rate of 1.4 mm/day for 6 days. TP508 was injected into as the following: Group 1, TP508 in saline; Group 2, in PPF/PLGA [poly(propylene fumarate)/poly(D,L-lactic-co-glycolic acid)] microparticles; and Group 3, dextran gel only. All the animals were killed 2 weeks after lengthening. On radiographies, more bone was formed in the two TP508-treated groups at first and secnd week postlengthening than that of the control Group 3. Microcomputed tomography (microCT) at 2 weeks indicated that the most advanced bone formation and remodeling was seen in Group 2. The mean volumetric BMD of the regenerates was significantly higher in the TP508 treated groups compared to the control group (p < 0.05). Histological evaluations supported the radiographic and the microCT results. In conclusion, we have demonstrated that a single injection of small amount of TP508 (300 microg) at the end of lengthening phases has significantly enhanced bone consolidation process in a rabbit model of distraction osteogenesis. The delivery of TP508 in PPF/PLGA microparticles appears to lead to a better quality bone formation over the saline delivery, further examinations are needed to confirm if PPF/PLGA microparticles may be desirable drug delivery form in augmenting bone formation.

Silk implants for the healing of critical size bone defects

Bone (re)-generation and bone fixation strategies utilize biomaterial implants, which are gradually replaced by autologous tissues. Ideally, these biomaterials should be biodegradable, osteoconductive, and provide mechanical strength and integrity until newly formed host tissues can maintain function. Some protein-based biomaterials such as collagens are promising because of their biological similarities to natural proteins on bone surfaces. However, their use as bone implant materials is largely hampered by poor mechanical properties. In contrast, silks offer distinguishing mechanical properties that are tailorable, along with slow degradability to permit adequate time for remodeling. To assess the suitability of silk-based biomaterials as implants for bone healing, we explored the use of novel porous silk fibroin scaffolds as templates for the engineering of bone tissues starting from human bone marrow derived stem cells cultured under osteogenic conditions for up to 5 weeks. The slowly degrading protein matrix permitted adequate temporal control of hydroxyapatite deposition and resulted in the formation of a trabecular-like bone matrix in bioreactor studies. The organic and inorganic components of the engineered bone tissues resembled those of bone, as shown by gene expression analysis, biochemical assays, and X-ray diffractometry. Implantation of the tissue-engineered bone implants (grown in bioreactors for 5 weeks prior to implantation) into calvarial critical size defects in mice demonstrated the capacity of these systems to induce advanced bone formation within 5 weeks, whereas the implantation of stem cell loaded silk scaffolds, and scaffolds alone resulted in less bone formation. These results demonstrate the feasibility of silk-based implants with engineered bone for the (re-)generation of bone tissues and expand the class of protein-based bone-implant materials with a mechanically stable and durable option.

Enhancement of bone formation in rat calvarial bone defects using low-level laser therapy

Objective To evaluate the effect of low-level laser therapy (LLLT), using a GaAlAs diode laser device, on bone healing and growth in rat calvarial bone defects. Study design An animal trial of 4 weeks' duration was conducted using a randomized blind, placebo-controlled design. Standardized round osseous defects of 2.7 mm diameter were made in each parietal bone of 20 rats (n = 40 defects). The animals were randomly divided into an experimental and a control group of 10 animals each. In the experimental group, a GaAlAs diode laser was applied immediately after surgery and then daily for 6 consecutive days. The control group received the same handling and treatment, but with the laser turned off. Five rats from each group were killed on day 14 and the remainder on day 28 postoperatively. From each animal, tissue samples from one defect were prepared for histochemistry and samples from the contralateral defect for histology. Levels of calcium, phosphorus, and protein were determined by using atomic absorption spectrometry, colorimetry, and photometry, respectively. Student t-test and Mann-Whitney were used for statistical analyses. Results At both time points the tissue samples from the experimental animals contained significantly more calcium, phosphorus, and protein than the controls. Similarly, histological analyses disclosed more pronounced angiogenesis and connective tissue formation, and more advanced bone formation in the experimental group than in the controls. Conclusion LLLT may enhance bone formation in rat calvarial bone defects.

A thyrotoxic skeletal phenotype of advanced bone formation in mice with resistance to thyroid hormone.

Thyroid hormone (T3) regulates bone turnover and mineralization in adults and is essential for skeletal development during childhood. Hyperthyroidism is an established risk factor for osteoporosis. Nevertheless, T3 actions in bone remain poorly understood. Patients with resistance to thyroid hormone, due to mutations of the T3-receptor beta (TRbeta) gene, display variable phenotypic abnormalities, particularly in the skeleton. To investigate the actions of T3 during bone development, we characterized the skeleton in TRbetaPV mutant mice. TRbetaPV mice harbor a targeted resistance to thyroid hormone mutation in TRbeta and recapitulate the human condition. A severe phenotype, which includes shortened body length, was evident in homozygous TRbetaPV/PV animals. Accelerated growth in utero was associated with advanced endochondral and intramembranous ossification. Advanced bone formation resulted in postnatal growth retardation, premature quiescence of the growth plates, and shortened bone length, together with increased bone mineralization and craniosynostosis. In situ hybridization demonstrated increased expression of fibroblast growth factor receptor-1, a T3-regulated gene in bone, in TRbetaPV/PV perichondrium, growth plate chondrocytes, and osteoblasts. Thus, the skeleton in TRbetaPV/PV mice is thyrotoxic and displays phenotypic features typical of juvenile hyperthyroidism.

Nanoparticulate Hydroxyapatite Enhances the Bioactivity of a Resorbable Bone Graft

ABSTRACTA nanoparticulate-hydroxyapatite filler augmented osteointegration within a resorbable polymer based bone graft substitute designed for orthopaedic and periodontal applications. The unsaturated polyester poly(propylene glycol-co-fumaric acid) (PPF), was used to prepare the bone graft substitute. The nanoparticulate-hydroxyapatite filler was examined in terms of biocompatibility, bony ingrowth and mechanical stability in a rat calvarial defect model. The nano-hydroxyapatite fillerwas compared against a commercially available micrometer-sized hydroxyapatite(HA) filler. Histological analysis indicated that remodeling of the newly formed bone was more advanced in the defect filled with the nano-hydroxyapatite augmented PPF. Mechanical evaluation showed a more rapid increase in stiffness of the nano-hydroxyapatite PPF. Implants of the nano-HA augmented PPF showed more advanced bone formation and recovery of mechanical properties, suggesting an improved biological response to the nano-sized particles.