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Controlling inflammation meanwhile facilitating tissue regeneration has been considered as a promising strategy to treat inflammatory bone defect. Herein, we describe the synthesis of a bio-sensitive poly(lactic-co-glycolic acid)/mesoporous silica nanocarriers core-shell porous microsphere (PLGA/MSNs-PMS) encapsulated poly(L-lactic acid) (PLLA) spongy nanofibrous micro-scaffold as a new generation of therapeutic platform for effective reconstruction of bone defects caused by periodontal diseases. The PLGA/MSNs-PMS were designed as stimuli-responsive carriers for on-demand co-delivery of multiple biomolecules to provide proper physiological environment, while the multi-level (from macro-, micro- to nanometers) nanofibrous and porous structures in PLLA micro-scaffold were in charge of the reconstruction of ECM, which synergistically contribute to the enhancement of new tissue formation under inflammatory condition. After local injection into periodontal tissue, this construct could sequentially release bone growth factor (BMP-2) as well as anti-inflammatory drug (celecoxib) loaded MSNs in response to the over-expressed matrix metalloproteinases (MMP) in periodontal region. During alveolar bone regeneration induced by BMP-2 and ECM like structure, the MSNs would further deliver celecoxib in target cells to achieve inflammation inhibition, resulting in effective treatment of periodontal disease.

Human bone morphogenetic protein-2 (BMP2) plays an important role in the development of bone and cartilage, which functions as bone growth factor by interacting with its cognate type-I and type-II receptors (BMPR-I and BMPR-II) through conformational wrist epitope and linear knuckle epitope, respectively. Here, the intermolecular interaction between the BMP2 knuckle epitope and BMPR-II is investigated at structural level, from which a binding hotspot region is identified that is primarily responsible for the epitope recognition by BMPR-II. A KEH peptide covering the hotspot region exhibits a weak binding affinity (Kd = 78.6 ± 13.5 µM) to BMPR-II and a moderate osteogenic activity on human BMSCs (ALP = 135 ± 17% and 164 ± 21% at peptide concentrations 0.01 and 0.1 µg/ml, respectively). Quantitative structure-activity relationship (QSAR) is used to guide the rational optimization of KEH peptide. The strategy focuses on systematic design of the peptide’s non-key residues to derive a series of its promising mutants, which are then evaluated rigorously by using dynamics simulation and energetic analysis. Consequently, totally ten designed peptide mutants are tested in vitro at molecular and cellular levels; most of them are determined to exhibit an increased or comparable affinity/activity relative to the native KEH peptide and knuckle epitope. In particular, the KEH-p7 peptide is found to have a satisfactory profile with Kd = 9.6 ± 1.2 µM and ALP = 178 ± 24% and 235 ± 32% at peptide concentrations 0.01 and 0.1 µg/ml, respectively. Structural analysis reveals a complicated noncovalent network of hydrogen bonds, hydrophobic forces and stacking interactions across the complex interface of BMPR-II with KEH-p7, conferring both stability and specificity to the receptor–peptide interaction.

A novel PTH-derived peptide, PTHdP, including a repetitive aspartic acid sequence at the C-terminal and phosphorylated serine at the N-terminal has been previously designed. To evaluate its potential as a bone growth factor for bone tissue engineering, true bone ceramics incorporated with nanohydroxyapatite coating and chitosan (CH/TBC) was developed as a desired three-dimensional porous delivery carrier in this study. In vitro results showed that PTHdP could be loaded with high-efficiency and subsequently released in a controlled and sustained manner from CH/TBC. Bioactivity of released PTHdP was retained and able to exert a significant effect on promoting or inhibiting osteogenesis actions when exposed intermittently or continuously, respectively, for MC3T3-E1 cell culture. As evaluated in a critical-size rabbit radial defect model by radiographic and histological examination, the combination of CH/TBC scaffolds with PTHdP exhibited a remarkably stronger capacity to stimulate new bone formation than control and pure CH/TBC groups. These results indicated the novel PTHdP peptide achieved high affinity to bone mineral without interference in bioactivity, and local delivery of PTHdP from apatite materials could be a promising alternative for future bone tissue engineering.

Lactoferrin (LF) is a basic glycoprotein and a dominant functional component from the whey protein in bovine milk. As a novel bone growth factor, it can fulfill its biological functions of preventing osteoporosis by regulating the growth and metabolic processes of bone. However, it has not been clarified that LF plays a role of osteogenesis in a form of molecular fragments after enzymatic digestion. In this study, a novel peptide with osteoblast proliferation activity, ENLPEKADRDQYEL, was identified using UPLC-Q-TOF-MS/MS and Mascot analysis. The mechanism of promoting proliferation of osteoblast activity was also analyzed by molecular docking. Results demonstrated that ENLPEKADRDQYEL can significantly promote the proliferation of osteoblasts. The main interaction forces of ENLPEKADRDQYEL with epidermal growth factor receptor (EGFR) were the hydrophobic and hydrogen bonding. ENLPEKADRDQYEL had similar target domain (Lys13-Leu14-Thr15-Gln16-Leu98-Ser99-Ser418) with the key structure of EGFR compared with epidermal growth factor (EGF). This work established a theoretical foundation for the peptide from lactoferrin used as a functional component in functional dairy products.

Lactoferrin (LF) has been recently recognized as a promising new novel bone growth factor for the beneficial effects on bone cells and promotion of bone growth. Currently, it has been attracted wide attention in bone regeneration as functional food additives or a potential bioactive protein in bone tissue engineering. The present study investigated the possibility that hydroxyapatite (HAP) particles, a widely used bone substitute material for high biocompatibility and osteoconductivity, functionalized with lactoferrin as a composite material are applied to bone tissue engineering. Two kinds of hydroxyapatite samples with different sizes, including nanorods and microspheres particles, were functionalized with lactoferrin molecules, respectively. A detailed characterization of as-prepared HAP-LF complex is presented, combining thermal gravimetric analysis (TGA) and Fourier Transform Infrared Spectroscopy (FT-IR). Zeta potential and the analysis of electrostatic surface potential of lactoferrin were carried to reveal the mechanism of adsorption. The effects of HAP-LF complex on MC3T3-E1 osteoblast proliferation and morphology were systematically evaluated at different culture time. Interestingly, results showed that cell viability of HAP-LF group was significantly higher than HAP group indicating that the HAP-LF can improve the biocompatibility of HAP, which mainly originated from a combination of HAP-LF interaction. These results indicated that hydroxyapatite particles can work as a controlled releasing carrier of lactoferrin successfully, and lactoferrin showed better potentiality on using in the field of bone regeneration by coupling with hydroxyapatite. This study would provide a new biomaterial and might offer a new insight for enhancement of bone regeneration.

Osteoporosis is a disease of aging, characterized by a decrease in bone quality and a reduction in bone strength. Promoting the activity of osteoblasts is a useful strategy for combating the progression of osteoporosis. As a novel bone growth factor, lactoferrin plays a role in the anabolic activity in bone by inducing the proliferation and differentiation of osteoblasts and inhibiting the formation of osteoclasts. However, potential peptides with osteogenic activity from lactoferrin have not been identified. In the present study, a peptide with osteogenic activity-LFP-C, fragment residues 624 to 632, derived from lactoferrin hydrolysates-was identified using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and screened using molecular docking analysis. The LFP-C peptide significantly increased the proliferation of mouse cell line MC3T3-E1 and had a promoting effect on alkaline phosphatase activity and calcium deposition. Moreover, LFP-C increased the proportion of osteoblasts in the G2 and M phases. The osteogenic mechanism of LFP-C was also studied by molecular docking. We found that LFP-C could bind to the key domain (Lys13-Thr15-Gln16-Leu17-Gly18-Asp22) of epidermal growth factor receptor, a vital receptor tyrosine kinase that leads to the activation of the mitogen-activated protein kinase pathway. The main interaction forces were interpolated charge, hydrophobicity, and hydrogen bonding. Results indicated that LFP-C may play an osteogenic role in a similar way to lactoferrin, by promoting the proliferation and differentiation of osteoblasts. The findings of this in vitro experiment also demonstrated that the molecular docking method could play a role in the screening process; this in silico approach allowed for faster and cheaper identification of a promising bioactive component.

Introduction According to neurohormonal theory, prenatal androgens are key determinants of sexual orientation. As a reputed marker for prenatal androgens, the 2D:4D finger length ratio has been used in more than a dozen studies to test the hypothesis that prenatal androgens influence sexual orientation. Findings have been very inconsistent. Aim The present study sought to retest the hypothesis that 2D:4D and sexual orientation are related is a manner consistent with neurohormonal theory. Methods A 2D:4D measure (of the right hand) along with four additional somatic markers of androgen exposure (height, physical strength, muscularity, and athletic ability) with samples of college students in Malaysia (N = 2,058) and the United States (N = 2,511). The five androgen measures were factor analyzed, resulting in a two-factor solution: Factor 1 consisted of strength, muscularity, and athletic ability (the muscular coordination factor), and Factor 2 was comprised of the r2D:4D and adult height (the bone growth factor). Main Outcome Measures Sexual orientation was measured by asking each respondent the extent to which they were sexually attracted to males and the extent to which they were sexually attracted to females, both on 11-point scales. Results When the countries and sexes were analyzed separately, neither the r2D:4D measure nor Factor 2 correlated with sexual orientation to significant degrees. Instead, it was the muscular coordination factor that correlated the best. Support was found for the hypothesis that prenatal androgens influence sexual orientation, but the nature of these influences was more complex than neurohormonal theory predicted. Conclusion A modified theory is needed and presented to accommodate the results from this study.

Osteogenic suppressors such as Sclerostin not only regulate skeletal development and regeneration but also serve as anti-osteoporosis drug targets. However, very few druggable suppressors have been identified due to limited understanding of the molecular mechanisms governing osteogenesis. Here, we show that fibroblast activation protein (Fap), a serine protease inhibited by the bone growth factor Osteolectin, is an osteogenic suppressor. Genetic deletion of Fap significantly ameliorates limb trabecular bone loss during aging. Pharmacological inhibition of Fap significantly promotes bone formation and inhibits bone resorption in wild-type mice by differentially regulating canonical Wnt and nuclear factor κB (NF-κB) pathways. Pharmacological inhibition of Fap promotes osteoblast differentiation, inhibits osteoclast differentiation, and significantly attenuates osteoporosis in ovariectomized mice. Epistasis analyses in zebrafish show that Osteolectin functions as an endogenous inhibitor of Fap to promote vertebrae mineralization. Taken together, we identify Fap as an important osteogenic suppressor and a potential drug target to treat osteoporosis.

Ti-based implants sometimes fail to integrate with surrounding bone tissue due to insufficiency of new bone formation and surface bonding. To overcome this problem, this research focused on establishing a sustained bone growth factor delivery system by applying anodized TiO2 nanotube arrays and PLGA film on the titanium implant surface. TiO2 nanotube arrays were made by anodic oxidation method, and were then filled with rhBMP2 by vacuum freeze-drying. Next, PLGA was deposition on the surface of this material. The designed system was characterized, pharmacokinetic release rate of rhBMP2 was determined. Adhesion, proliferation, and differentiation activity of osteoblasts cultured on the new surfaces and traditional titanium surfaced were compared. SEM showed that a surface of TiO2 nanotube arrays were successfully generated. PLGA membranes of 50 nm, 250 nm, 800 nm thickness were successfully deposited on the surfaces of TiO2 nanotube layers by using 1%, 3%, 10% PLGA solutions. PLGA film of 250 nm thickness showed ideally controlled release of rhBMP2, lasting for 4 weeks. Furthermore, 250 nm thickness PLGA film improved osteoblast adhesion, proliferation, and levels of alkaline phosphatase. In conclusion, the PLGA film / TiO2 nanotube growth factor delivery system can effectively sustain the release of rhBMP-2, and promote proliferation and differentiation of MC3T3-E1 osteoblasts.

As interface design of autotransportation materials and bioactive ceramics by supersonic treatment, human teeth-originated granules and commercial hydroxyapatite (HAp) were partially dissolved in different acid solutions, such as electrolyzed water and HNO3aqueous solution to control bio-absorption, adsorption-release of bone growth factor and anti-bacterial characteristics. Human teeth were pulverized with cooling, dissolved in strongly acidic electrolyzed water (pH 2.6-3.1) or 2.0% HNO3solution to obtain demineralized dentin matrix granules. For supersonic or stirring demineralization in the acidic electrolyzed water, dissolution efficiencies (DE) were 3-12% and dentinal tubules with 1-2μm were found, while for supersonic demineralization in the HNO3solution, DE was attained to the highest value of 86%. When HAp was dissolved with stirring in the acidic electrolyzed water, DE of porous ceramics was lower than that of spherical particles due to smaller specific surface area and good crystallinity. For supersonic dissolution of porous ceramics in the acidic electrolyzed water, DE was 30% and extension of grain boundary and micro-crack were observed. Concerning supersonic irradiation of the electolyzed water to parietal bone in rats by using ultrasonic scaler, enlargement and propagation of micro-crack were recognized on the hard tissues.

Osteoporosis is a systemic disease characterized by bone loss and bone tissue micro-structural damage, leading to bone fragility and an increased risk of bone fractures. The main purpose of osteoporosis treatment is to prevent the occurrence of fracture, but current drugs rarely achieve this goal with specific and notable therapeutic effects. There are mainly three types of drug used for osteoporosis treatment: bone resorption inhibitors is the largest class, it can increase bone mineral density through inhibiting bone resorption; bone anabolic agents can stimulate bone formation directly and rebuild bone tissue; bone mineralizers is the basic medicine of preventing and curing osteoporosis, and it includes calcium and vitamin D. However, with the development of scientific research, some new treatment concepts and strategies are proposed, and targeted therapy has become the development tendency of drugs in the future. Currently, various bone growth factor preparations (such as Denosumab, Sclerostin antibody, etc) have been gradually developed, which are based on studies of RANKL signaling pathway and Wnt signaling pathway, and their effect is better. In addition, as an important regulatory pathway, BMP signaling pathway plays a prominent role in stimulating bone formation. Regulatory proteins of this pathway have many potential targets, such as Smurf1, CKIP-1. It is worth discussing in the research and development of new drugs for osteoporosis treatment.

β-Tricalcium phosphate (β-TCP) with three different particle size ranges was used to study the effects of particle size and surface area on protein adsorption and release. Polycaprolactone (PCL) coating was applied on the particle systems to investigate its effect on particulate system properties from both structural and application aspects. The maximum loading of 27 mg/g was achieved for 100 nm particles. Bovine serum albumin (BSA) loading amount was controlled by varying the BSA loading solution concentration, as well as the sample powder's surface area. Increasing the surface area of the delivery powder significantly increased loading and release yield. Unlike the samples with low surface area, the lowest particle size samples showed sigmoidal release profile. This indicated that release was governed by different mechanisms for particles with different sizes. While the majority of samples showed no more than 50% release, the 550 nm particles demonstrated 100% release. PCL coating showed no significant ability to attenuate burst release in PBS. However, it led to a steadier release profile as compared to the bare TCP particles. FTIR analysis also proved that the secondary structure of BSA did not change significantly during the adsorption; however, minor denaturation was found during the release. The same results were found when PCL coating was applied on the TCP particles. We envision potential use of TCP and TCP + PCL systems in bone growth factor or orthopedic drug delivery applications in future bone tissue engineering application.

A kind of PLGA microspheres was prepared with bovine serum albumin (BSA) as the model drug and poly (lactide-co-glycolide) as the matrix. The polylactic acid/hydroxyapatite (PLA/HA) scaffold was manufactured through 3D printing technology. Then the PLGA microspheres were composited in the scaffold. It was also explored about the feasibility of skeletal scaffolds loaded with bone growth factor. The BSA loading PLGA microspheres were prepared by W/O/W method and the scaffolds were prepared by 3D-printing using PLA and HA as raw materials. The composite scaffold was fabricated by adsorbing the microspheres/ethanol suspension into scaffolds under negative pressure. The cell-adhesion ability, hydrophilicity, scaffold morphology, release properties and biocompatibility of the composite scaffold were characterized, respectively. The results show no burst release of BSA from the PLGA microspheres at beginning stage and sustained longer than 35 days. Drug-loading rate of microspheres was 0.64%. PLA/HA scaffold shows enhanced hydrophilicity as well as excellent cell compatibility and cell adhesion property. SEM images show PLGA microspheres were successfully absorbed in PLA/HA scaffold. MTT experiments of the composite scaffold show non cytotoxic and its cell relative proliferation rate is up to 88.37%. These studies show the feasibility of skeletal scaffolds loaded with bone growth factor. Through low-temperature rapid prototyping technology, the long-effective bioactive bone scaffold can be prepared and have a well application prospect.

Socially responsible technologies are designed while taking into consideration the socioeconomic, geopolitical and environmental limitations of regions in which they will be implemented. In the medical context, this involves making therapeutic platforms more accessible and affordable to patients in poor regions of the world wherein a given disease is endemic. This often necessitates going against the reigning trend of making therapeutic nanoparticles ever more structurally complex and expensive. However, studies aimed at simplifying materials and formulations while maintaining the functionality and therapeutic response of their more complex counterparts seldom provoke a significant interest in the scientific community. In this review we demonstrate that such compositional simplifications are meaningful when it comes to the design of a solution for osteomyelitis, a disease that is in its natural, non-postoperative form particularly prevalent in the underdeveloped parts of the world wherein poverty, poor sanitary conditions, and chronically compromised defense lines of the immune system are the norm. We show that calcium phosphate nanoparticles, which are inexpensive to make, could be chemically designed to possess the same functionality as a hypothetic mixture additionally composed of: (a) a bone growth factor; (b) an antibiotic for prophylactic or anti-infective purposes; (c) a bisphosphonate as an antiresorptive compound; (d) a viral vector to enable the intracellular delivery of therapeutics; (e) a luminescent dye; (f) a radiographic component; (g) an imaging contrast agent; (h) a magnetic domain; and (i) polymers as viscous components enabling the injectability of the material and acting as carriers for the sustained release of a drug. In particular, calcium phosphates could: (a) produce tunable drug release profiles; (b) take the form of viscous and injectable, self-setting pastes; (c) be naturally osteo-inductive and inhibitory for osteoclastogenesis; (d) intracellularly deliver bioactive compounds; (e) accommodate an array of functional ions; (f) be processed into macroporous constructs for tissue engineering; and (g) be naturally antimicrobial. All in all, we see in calcium phosphates the presence of a protean nature whose therapeutic potentials have been barely tapped into.

Background: Reconstruction of the anterior cruciate ligament (ACL) involves use of semintendinosis and gracilis tendons graft that is transplanted into bone tunnels at the femoral and tibial insertion sites and the sites and the bone tendon interface is a weak link in the early healing period due to slow rate of healing. We hypothesized that an addition of bone growth factor like Sadat-Habdan mesenchymal stimulating peptide (SHMSP) could enhance bone tendon healing rate so that re-rupture of the tendon does not take place. Methodology: Twenty skeletally mature rabbits underwent ACL reconstruction of the right knee. In 10 of the rabbits at the site of the tendon-graft 5 mg/kg body weight of SHMSP was put in the bone tunnel. In 10 other animals, nothing was added. At eight and 12 weeks 5 animals from each group were sacrificed. The tendon-graft site was harvested and sent for histopathological examination to assess the healing at the tendon-bone graft to the tibial tunnel. Results: There were no deaths in both the groups. One rabbit of the control group developed an infection. In all the animals of the study group from 4 weeks onward showed bone formation, wherein the control group only granulation tissue was observed. By 8 weeks in the study group, the canal was totally obliterated with the new bone formation which extended onto the periosteal area. In the control, there was minimal change in the formation of the new bone formation. Conclusion: Addition of a growth factor like SHMSP would enhance the osteo-integration of the tendon-graft in the bony tunnel after ACL reconstruction in vivo.

A series biodegradable poly (γ-glutamic acid)/beta-tricalcium phosphate (γ-PGA/TCP) nanocomposites were prepared which were composed of poly-γ-glutamic acid polymerized in situ with β-tricalcium phosphate and physiochemically characterized as bone graft substitutes. The particle size via dynamic light scattering, the direct morphological characterization via transmission electron microscopy and field emission scanning electron microscope, which showed that γ-PGA and β-TCP were combined compactly at 80℃, and the γ-PGA/TCP nanocomposites had homogenous and nano-sized grains with narrow particle size distributions. The water uptake and retention abilities, in vitro degradation properties, cytotoxicity in the simulated medium, and protein release of these novel γ-PGA/TCP composites were investigated. Cell proliferation in composites was nearly twice than β-TCP when checked in vitro using MC3T3 cell line. We also envision the potential use of γ-PGA/TCP systems in bone growth factor or orthopedic drug delivery applications in future bone tissue engineering applications. These observations suggest that the γ-PGA/TCP are novel nanocomposites with great potential for application in the field of bone tissue engineering.

Some evidence suggests that prenatal androgens influence both handedness and sexual orientation. This study sought to clarify how androgens, handedness, and sexual orientation are interrelated.Data were obtained from large samples of students enrolled at universities in Malaysia and the US, including self-reported information on handedness, sexual orientation, and five somatic markers of prenatal androgen exposure (2D:4D, height, strength, muscularity, and athletic ability). Factor analysis of these somatic markers yielded two factors: a muscular coordination and a bone growth factor.In women, but not in men, ambidextrousness was more prevalent among those with homosexual tendencies. Modest and often complex associations were found between the androgen factors and handedness. Clear links between the androgen factors and sexual orientation were found, especially for muscular coordination. For males and females, intermediate sex-typical androgen exposure was associated with heterosexual preferences.Ambidextrousness appears to be somewhat more common among females with homosexual tendencies, but left-handedness is nearly as strongly associated with heterosexual preferences, particularly in males, as is right-handedness. Factors indicative of prenatal androgen exposure are associated with sexual orientation in theoretically predictable ways, especially for muscular coordination, but associations between prenatal androgens and handedness are complex.

Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS-5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung do not generate effects as great as HS5. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively supports bone formation, and suggest its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2.

Nonunion is a common problem in Orthopedic Surgery. In the recent years alternatives to the standard surgical procedures were tested clinically and in vitro. Extracorporeal shock wave therapy (ESWT) showed promising results in both settings. We hypothesized that in target tissue cells from nonunions like fibroblasts and osteoblasts ESWT increases the release of bone growth factors.Fibroblasts and osteoblasts were suspended in 3 ml cryotubes and subjected to 250/500 shock waves at 25 kV using an experimental electrohydraulic lithotripter. After ESWT, cell viability was determined and cells were seeded at 1 × 10(5) cells in 12 well plates. After 24, 48, and 72 h cell number was determined and supernatant was frozen. The levels of growth factors FGF-2 and TGF-β(1) were examined using ELISA. A control group was treated equally without receiving ESWT.After 24 h there was a significant increase in FGF-2 levels (p 0.05) with significant correlation to the number of impulses (p 0.05) observed. TGF-β(1) showed a time-dependent increase with a peak at 48 h which was not significantly different from the control group.FGF-2, an important growth factor in new bone formation, was shown to be produced by human fibroblasts and osteoblasts after treatment with ESWT. These findings demonstrate that ESWT is able to cause bone healing through a molecular way by inducing growth factor synthesis.

Failure of bone and joint implants has been attributed mainly to poor bonding of the implant to bone tissue, and to bacterial infection. The probability of successful osseointegration or implant infection depends on the race for the surface between tissue cells and bacteria. One promising strategy to enhance tissue integration is to develop a selective biointeractive surface that increases bone cell (osteoblast) function while decreasing bacterial adhesion. In this in vitro study, the surface of titanium alloy substrates was first functionalized by covalently grafted oxidized dextran, which is known to have activity against bacterial adhesion. Bone morphogenetic protein-2 (BMP-2) was then covalently linked to dextran-grafted surfaces through a chemical conjugation process. The composition and properties of the surface were investigated by X-ray photoelectron spectroscopy and by measuring the surface density of BMP-2 using an enzyme-linked immunosorbent assay. Bacterial adhesion was assayed with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial adhesion on both the dextran and dextran-BMP-2-functionalized surfaces was significantly decreased compared to that on the pristine substrates. Further, the dextran-BMP-2 modified substrates with a surface protein density of >50 ng/cm(2) or higher significantly promoted osteoblast spreading, alkaline phosphatase activity, and calcium mineral deposition. Thus, the results from this study suggest that surface grafting of dextran in conjunction with the bone growth factor BMP-2 on metal surfaces can enhance tissue integration of implants through the dual functions of reducing bacterial adhesion and promoting osteoblast functions.

Structured Abstract Authors – Arosarena O, Collins W Objective – To compare mandibular bone regeneration with bone morphogenetic proteins-2 and -4 (BMP-2 and -4) at varying doses. Study Design – Defects were created in the left hemi-mandibles of 82 Sprague–Dawley rats. The defects were filled with a hyaluronic acid polymer loaded with 0.01, 0.1, 1, or 10 μg of BMP-2 or -4. Control groups consisted of animals with unfilled defects, or with defects filled with the hyaluronic acid sponges loaded with growth factor dilution buffer. Animals were killed after 8 weeks, and the hemi-mandibles were analyzed histologically using stereologic techniques. Results – Mandibles implanted with carriers containing 10 μg of BMP-2 or -4 differed significantly from controls in terms of new bone area (p = 0.01 and p = 0.0001, respectively). Marrow space development occurred in a dose-dependent fashion (p

Currently there is no consensus on the impact of dietary protein on calcium and bone metabolism. This study was conducted to examine the effect of increasing protein intake on urinary calcium excretion and to compare circulating levels of IGF-I and biochemical markers of bone turnover in healthy older men and women who consumed either a high or a low protein food supplement for 9 wk. Thirty-two subjects with usual protein intakes of less than 0.85 g/kg.d were randomly assigned to daily high (0.75 g/kg) or low (0.04 g/kg) protein supplement groups. Isocaloric diets were maintained by advising subjects to reduce their intake of carbohydrates. Selected biochemical measurements were made at baseline and on d 35 and either d 49 or 63. Changes in urinary calcium excretion in the two groups did not differ significantly over the course of the study. The high protein group had significantly higher levels of serum IGF-I (P = 0.008) and lower levels of urinary N-telopeptide (P = 0.038) over the period of d 35-49 or 63. We conclude that increasing protein intake from 0.78 to 1.55 g/kg.d with meat supplements in combination with reducing carbohydrate intake did not alter urine calcium excretion, but was associated with higher circulating levels of IGF-I, a bone growth factor, and lowered levels of urinary N-telopeptide, a marker of bone resorption. In contrast to the widely held belief that increased protein intake results in calcium wasting, meat supplements, when exchanged isocalorically for carbohydrates, may have a favorable impact on the skeleton in healthy older men and women.

Study design Prospective animal and human clinical pilot trial. Objectives The purpose of this study was to determine and test the dose of Ne-Osteo growth factor extract and carrier required for consistent radiographic bone induction in humans. Summary of background data Preclinical studies have demonstrated that Ne-Osteo, an extract-containing bone morphogenetic proteins, was successful at generating spine fusion in rabbits and rhesus monkeys. Consistent fusions have yet to be achieved in nonhuman primates and humans. Methods Adult rhesus monkeys underwent single-level posterolateral intertransverse lumbar arthrodesis with either 3.0 mg (N = 4), 5.0 mg (N = 4), 12.5 mg (N = 4), or 25 mg (N = 4) of Ne-Osteo per side. Animals were killed after 24 weeks. In the human clinical trial, 22 patients (18 females, 4 males) had lumbar spinal stenosis and/or spondylolisthesis requiring spine arthrodesis. To minimize patient risk of nonunion, patients received autogenous bone graft from the posterior iliac crest on one side and Ne-Osteo growth factor on the other. The dose was 12.5 mg, 25 or 50 mg, or 25 mg Ne-Osteo per side performed in the three phases, respectively. Results Three of four monkeys that received 12.5 mg Ne-Osteo per side and four of four that received 25 mg per side achieved solid fusions. In phase I of the human clinical trial, two of six patients showed radiographic bone induction (plain radiograph, CT scans-blindly evaluated) on the Ne-Osteo side (12.5-mg dose). In phase II, both sides were graded as fused in five of six patients. Although graded as fused, the 6-month scans demonstrated a ring of new bone with the center filling in slower (12-24 mo) than was predicted by nonhuman primate studies. As a result, phase III carrier was designed to have a more porous/open early fusion mass than with the dense DBM paste (used in phase I and II) by mixing in local bone or cancellous allograft chips. Results using the 25- and 50-mg doses were the same, so 25 mg was used in phase III. In phase III, 9 of 10 autograft were fused by 12 months. Five of five patients with Ne-Osteo plus local bone and four of five with allograft chips were fused by 6 months. The one patient in this group that did not heal on either the autograft or the Ne-Osteo side was a smoker. Conclusions A graft composite of Ne-Osteo bone growth factor with human DBM with or without cancellous allograft or local bone autograft was capable of achieving a contiguous spine fusion mass in 15 of 16 patients at a dose of at least 25 mg per side. This result was comparable with the results using iliac crest autograft (94%) in this side-by-side model. These results warrant confirmation in a definitive trial using Ne-Osteo on both sides of the spine and thus avoiding the need for iliac crest bone graft harvest.

During minimally invasive orthopedic surgeries, surgical intervention is required at two stages; to attain hemostasis and subsequently to implant the bone graft or its substitute. There is an apparent need for a material which can simultaneously control bone bleeding and provide support for bone repair. In this work, a moldable putty, which can be applied to bone defects (usually irregular in shape), was developed to address this need. It comprises of a hemostatic factor thrombin, osteoinductive "yolk-shell" particles containing bone growth factor (BMP-2), and an osteoconductive component hydroxyapatite. The yolk shell particles allowed controlled release of BMP-2 and showed significantly enhanced osteogenic differentiation of C2C12 (mouse myoblast) cells as demonstrated by increased alkaline phosphatase (ALP) activity and relative gene expressions of osteogenic differentiation markers. These particles were assembled into a moldable putty by mixing them with hydroxyapatite and silk fibroin solution (binding agent) supplemented with thrombin. The putty showed non-cytotoxicity, hemostatic ability, sustained release of BMP-2 and induced increased mineralization in C2C12 cells. This putty, if applied to bone defects during surgeries, may help attain hemostasis and may enhance bone repair by providing sustained release of bone growth factors.

We hypothesized that an exogenous bone growth factor could augment healing of a tendon graft in a bone tunnel in a rabbit anterior cruciate ligament-reconstruction model. Seventy rabbits underwent bilateral anterior cruciate ligament reconstructions with a semitendinosus tendon graft. One limb received a collagen sponge carrier vehicle containing a mixture of bone-derived proteins while the contralateral limb was treated with either no sponge or a sponge without bone-derived proteins. The reconstruction was evaluated at 2, 4, or 8 weeks with histologic, biomechanical, and magnetic resonance imaging analysis. Histologic analysis demonstrated that specimens treated with bone-derived proteins had a more consistent, dense interface tissue and closer apposition of new bone to the graft, with occasional formation of a fibrocartilaginous interface, when compared with control specimens. The treated specimens had significantly higher load-to-failure rates than did control specimens. Treatment with bone-derived proteins resulted in an average increase in tensile strength of 65%. The treated specimens were stronger than control specimens at each time point, but the difference was greatest at 8 weeks. On the basis of signal characteristics and new bone formation, magnetic resonance imaging was useful for predicting which limb was treated, the site of failure, and the limbs with higher load-to-failure values. This study demonstrates the potential for augmenting tendon healing in an intraarticular bone tunnel using an osteoinductive growth factor.

In order to develop bone substitutes, the design of biomaterials like calcium phosphate ceramic loaded with bone growth factor are of great interest. However, it is necessary to control the amount of growth factor adsorbed onto ceramics and the kinetics of its release. Radiolabeling of insulin-like growth factor-I (IGF-I) with 125-iodine ([(125)I]-IGF-I) and its adsorption onto porous tricalcium phosphate (TCP) cylinders enabled us to establish the time-adsorption and time-release curves using various concentrations of IGF-I. The adsorption curve increased rapidly and then flattened out at 72 h; 90% of the maximum was already reached at 24 h; and 20% of the adsorbed IGF-I was released in water within 4 days. In human serum the release was faster at 82% within 4 days. In vivo evaluation on an animal model was then performed. Rabbits' bilateral femoral cylindrical bone defects were filled with the TCP cylinders, which were either carrying IGF-I or implanted alone as a control in each rabbit. Bone turnover and ceramic resorption were stimulated by IGF-I loaded TCP according to standard radiography, dual-energy X-ray absorptiometry, histology, and histomorphometry.

Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-δ (C/EBPδ) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPδ and C/EBPβ expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPδ and C/EBPβ on IGF-I expression by osteoblasts.

In this tutorial review we present the process of the development of functional implants using mesoporous silica. The different steps from chemical synthesis and physicochemical characterization followed by in vitro testing in cell culture assays to clinically relevant in vivo animal studies are examined. Since the end of the 1990s, mesoporous silicas have been considered as biomaterials. Numerous investigations have demonstrated their non-toxic and biocompatible properties. These qualities in combination with the unique properties of high surface area and pore volume, uniform and tunable pore sizes and chemical modifiability are the reasons for the great scientific interest in this field. Here we show that besides bulk materials or mesoporous silica nanoparticles, mesoporous silica films are highly promising as coatings on medical prostheses or implants. We report on the development of functionalized mesoporous silica materials specifically for middle ear applications. Middle ear prostheses are used to restore the sound transmission through this air-filled cavity when the small bones of the middle air (the ossicular chain) have been destroyed by disease or by accidents. In addition to optimal restoration of sound transmission, this technique bears several challenges, e.g. an ongoing bacterial infection or the displacement of the prosthesis due to insufficient fixation. To improve the healing process, a mesoporous silica coating was established on ceramic middle ear prostheses, which then served as a base for further functionalizations. For example, the bone growth factor BMP2 was locally attached to the coating in order to improve the fixation of the prosthesis by forming a bony connection to the remainder of the ear bones. Further, an implant-based local drug delivery system for the antibiotic ciprofloxacin was developed with the aim of fighting bacterial infections. Further possibilities using mesoporous silica nanoparticles as part of a composite on an implant are briefly discussed. © The Royal Society of Chemistry 2013.

The immunohistochemical expression and localization of some ossification-related factors, including a bone growth factor (bone morphogenetic protein, BMP), non-collagenous proteins (osteocalcin, BGP; bone sialoprotein, BSP), and type II collagen, were compared in union and non-union models of fracture healing in rats. In the union model, fracture healing was seen by day 25, whereas the non-union model did not show healing even on day 32. In both models, BMP, BGP, BSP, and type II collagen were expressed mainly in chondrocytes, with proliferating cell nuclear antigen (PCNA)-positive reaction in the soft callus. In the non-union model, the expression of BMP, BGP, BSP, type II collagen, and PCNA disappeared earlier than in the union model. In fracture healing, PCNA-positive cells expressed the ossification-related factors, these being essential for ossification.

Despite the potential for growth factor delivery strategies to promote orthopedic implant healing, there is a need for growth factor delivery methods that are controllable and amenable to clinical translation. We have developed a modular bone growth factor, herein termed "modular bone morphogenetic peptide (mBMP)", which was designed to efficiently bind to the surface of orthopedic implants and also stimulate new bone formation. The purpose of this study was to coat a hydroxyapatite-titanium implant with mBMP and evaluate bone healing across a bone-implant gap in the sheep femoral condyle. The mBMP molecules efficiently bound to a hydroxyapatite-titanium implant and 64% of the initially bound mBMP molecules were released in a sustained manner over 28 days. The results demonstrated that the mBMP-coated implant group had significantly more mineralized bone filling in the implant-bone gap than the control group in C-arm computed tomography (DynaCT) scanning (25% more), histological (35% more) and microradiographic images (50% more). Push-out stiffness of the mBMP group was nearly 40% greater than that of control group whereas peak force did not show a significant difference. The results of this study demonstrated that mBMP coated on a hydroxyapatite-titanium implant stimulates new bone formation and may be useful to improve implant fixation in total joint arthroplasty applications.

Fractures of the tibial shaft are likely to result in delayed union or non-union; 10–30% of these fractures will not heal properly and are commonly treated with autologous bone grafting. BMP-7 is a recombinant bone growth factor that can be applied locally as an alternative or in addition to autologous bone grafting, and this study aimed to compare the efficiency of the two procedures. From January 1995 to December 2002, 82 people with delayed union of a tibial fracture at least 4 months after primary stabilisation underwent autologous bone grafting. Successful healing was defined as radiological bony consolidation. Between May 2002 and June 2005, 26 similar cases were treated after the failure of the graft with local implantation of BMP-7, and were followed up for at least 1 year. Of the 82 people receiving autologous bone grafts only, 24 (28%) still had no signs of consolidation after 4 months and required revision surgery. Of the 26 people with BMP-7 implantation after failed graft, bony consolidation was seen after 4 months in 24 cases and only 2 (8%) required revision surgery. The BMP-7 group, although including more complicated cases, showed a significantly higher ( p = 0.025) success rate compared with the group that did not receive BMP-7.

Objectives/Hypothesis: Advances in bone repair have focused on the minimally-invasive delivery of tissue-engineered bone (TEB). A promising injectable biopolymer of chitosan and inorganic phosphates was seeded with mesenchymal stem cells (MSCs) and a bone growth factor (BMP-2), and evaluated in a rat calvarial critical size defect (CSD). Green fluorescent protein (GFP)-labeled MSCs are used to evaluate patterns of cell viability and proliferation. Study Design: Prospective, controlled trial in an animal model. Methods: In 30 male rats, 8-mm calvarial CSDs were created, and divided into five groups of six animals each. In the experimental groups, the defects were injected with either chitosan gel, gel loaded with MSCs (0.3 × 106 cells/defect), gel loaded with BMP-2 (2 μg/defect), or gel loaded with both MSC and BMP-2. In the control group, the defect was left untreated. At 4 weeks, in vivo microcomputed tomography (micro-CT) analysis was performed. At 8 weeks, calvarial specimens were examined by micro-CT, histology, and immunohistochemistry. Results: New areas of bone growth were seen in the defects of all treated animals. Micro-CT analysis revealed a significant (P < .001) time-dependent increase in the regeneration of bone volume and bone area in defects treated with gel/MSC/BMP-2 as compared to all other groups. Histological analysis confirmed this difference. GFP–labeled TEB was detected within the areas of new bone, indicating cell viability and contribution to new bone growth by the injected MSC. Conclusions: This study demonstrates that an injectable form of TEB using a chitosan gel, MSC, and BMP-2 can enhance bone formation in a rat calvarial CSD. Laryngoscope, 2010

Bone regeneration for the defects in revision surgery of joint replacement is an increasingly important issue. To repair bone defects, bone cell activation by growth factors using synthetic resorbable scaffold is a useful and safe option. We examine the efficiency of nanogel-crosslinking hydrogel as a novel synthetic scaffold for BMP to stimulate osteoblasts and to induce bone formation. Cholesterol-bearing pullulan nanogel-crosslinking hydrogel (CHPA/Hydrogel) was used to deliver BMP. The CHPA hydrogel pellets were implanted in vivo. Single implantation of CHPA/hydrogel containing low amounts of BMP induced osteoblastic activation and new bone formation in vivo. Furthermore, nanogel in a disc shape established recruitment of osteoblastic cells that vigorously formed bone to heal the calvarial defects, which did not heal spontaneously without it. In conclusion, CHPA/hydrogel serves as an efficient and versatile scaffold for the stimulation of osteoblasts to form bone and to repair defects via delivery of BMP.

In this study, bone marrow stromal cells (BMSCs) were differentiated on cyclic acetal composites containing hydroxyapatite (HA) particles (110 or 550 nm). These composites were evaluated for their role in influencing osteogenic signalling by encapsulated BMSCs. While a number of factors exert influence on osteogenic signalling during the production of an osteogenic matrix, we hypothesize that HA particles may upregulate bone growth factor expression due to enhanced BMSC adhesion. To this end, fluorescence-activated cell sorting (FACS) analysis was performed for the evaluation of BMSC surface marker expression after culture on two-dimensional (2D) cyclic acetal/HA composites. Three-dimensional (3D) composites were then fabricated by incorporating 110 or 550 nm HA particles at 5, 10 and 50 ng/ml concentrations. Bone growth factor molecules (TGFbeta1, FGF-2 and PDGFa), bone biomarker molecules (ALP, OC, OPN and OCN) and extracellular matrix-related molecules (FN, MMP-13, Dmp1 and aggrecan) were selected for evaluation of osteogenic signalling mechanisms when in presence of these composites. FACS results at day 0 demonstrated that BMSCs were a heterogeneous population with a small percentage of cells staining positive for CD29, CD90 and CD51/61, while staining negative for CD34 and CD45. At day 3, a significant enrichment of cells staining strongly for CD29, CD90 and CD51/61 was achieved. Gene expression patterns for bone growth factors and extracellular matrix molecules were found to be largely dependent upon the size of HA particles. Bone marker molecules, except OCN, had unaltered expression patterns in response to the varied size of HA particles. Overall, the results indicate that larger-sized HA particles upregulate PDGF and these groups were also associated with the most significant increase in osteodifferentiation markers, particularly ALP. Our results suggest that endogenous signalling is dependent upon material properties. Furthermore, we propose that studying gene expression patterns induced by the surrounding biomaterials environment is a fundamental step in the creation of engineered tissues.

We describe a phenotype-driven mutagenesis screen in which mice carrying a targeted mutation are bred with ENU-treated males in order to provide a sensitized system for detecting dominant modifier mutations. The presence of initial mutation renders the screening system more responsive to subtle changes in modifier genes that would not be penetrant in an otherwise wild type background. We utilized two mutant mouse models: 1) mice carrying a mutation in growth hormone releasing hormone receptor (Ghrhr) (denoted 'lit' allele, Ghrhr(lit)), which results in GH deficiency; and 2) mice lacking Smad2 gene, a signal transducer for TGF-beta, an important bone growth factor. The Smad2(-/-) mice are lethal and Ghrhr(lit/lit) mice are dwarf, but both Smad2(+/-) and Ghrhr(lit/)(+) mice exhibit normal growth. We injected 6-7 weeks old C57BL/6J male mice with ENU (100 mg/kg dose) and bred them with Ghrhr(lit/)(+) and Smad2(+/-) mice. The F1 mice with Ghrhr(lit/)(+) or Smad2(+/-) genotype were screened for growth and skeletal phenotypes. An outlier was identified as3 SD units different from wild type control (n=20-30). We screened about 100 F1 mice with Ghrhr(lit/)(+) and Smad2(+/-) genotypes and identified nine outliers. A backcross established heritability of three mutant lines in multiple generations. Among the phenotypic deviants, we have identified a mutant mouse with 30-40% reduced bone size. The magnitude of the bone size phenotype was amplified by the presence of one copy of the disrupted Ghrhr gene as determined by the 2-way ANOVA (p0.02 for interaction). Thus, a new mouse model has been established to identify a gene that interacts with GH signaling to regulate bone size. In addition, the sensitized screen also demonstrated higher recovery of skeletal phenotypes as compared to that obtained in the classical ENU screen in wild type mice. The discovery of mutants in a selected pathway will provide a valuable tool to not only to discover novel genes involved in a particular process but will also prove useful for the elucidation of the biology of that process.

Lactoferrin is an iron-binding glycoprotein that belongs to the transferrin family. It is present in breast milk, in epithelial secretions, and in the secondary granules of neutrophils. In healthy subjects lactoferrin circulates at concentrations of 2-7 x 10(-6) g/ml. Lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Recently, we have shown that lactoferrin can also promote bone growth. At physiological concentrations, lactoferrin potently stimulates the proliferation and differentiation of primary osteoblasts and also acts as a survival factor inhibiting apoptosis induced by serum withdrawal. Lactoferrin also affects osteoclast formation and, in murine bone marrow culture, lactoferrin potently inhibits osteoclastogenesis. In vivo, local injection of lactoferrin above the hemicalvaria of adult mice results in substantial increases in the dynamic histomorphometric indices of bone formation and bone area. The mitogenic effect of lactoferrin in osteoblast-like cells is mediated mainly through LRP1, a member of the family of low-density lipoprotein receptor-related proteins that are primarily known as endocytic receptors. Using confocal laser scanning microscopy, we demonstrated that fluorescently labeled lactoferrin is endocytosed and can be visualized in the cytoplasm of primary osteoblastic cells. Lactoferrin also induces activation of p42/44 MAPK signaling in primary osteoblasts, but the two pathways seem to operate independently as activation of MAPK signaling, but not endocytosis, is necessary for the mitogenic effect of lactoferrin. We conclude that lactoferrin may have a physiological role in bone growth and healing, and a potential therapeutic role as an anabolic factor in osteoporosis.

In an effort to decrease the number of problems associated with osteoporosis, the long-term goal of the present study is to design calcium phosphate-based nanoparticles that specifically attach to areas of low bone density and once attached, allow for the targeted release of bioactive agents that can quickly increase bone formation. Efforts are focused on nanoparticles of calcium phosphate-based materials since they are similar in size and chemistry to the major inorganic components of bone. As a first step in this research, the objective of the present study was to synthesize nanoparticles of crystalline hydroxyapatite (or HA) and amorphous calcium phosphate. Crystalline HA is stable under physiological fluids and, thus, will release embedded bioactive agents slowly. Alternatively, amorphous calcium phosphate is highly biodegradable and will, thus, release embedded bioactive agents quickly. A further objective of the present study was to functionalize such inorganic biodegradable materials with amino groups which would allow for the subsequent attachment of entities to direct such nanoparticles to osteoporotic bone and increased bone formation once attached. One promising approach to direct the nanoparticles to osteoporotic bone is to attach antibodies to pentosidine on the nanoparticles since pentosidine is present in higher amounts in osteoporotic compared to healthy bone. A promising approach to increase bone growth once nanoparticles attach to osteoporotic bone, is to embed nanoparticles with regions of the bone growth factor: bone morphogenic protein-2 (or BMP-2). Results of this study demonstrated the successful synthesis of both crystalline HA and amorphous calcium phosphate nanoparticles. Furthermore, results showed that these nanoparticles can be functionalized with versatile amino groups. In this manner, this study takes the first steps toward utilizing calcium phosphate based nanoparticles to reverse bone loss associated with osteoporosis.

To investigate the ability of a bone growth factor mixture and bone marrow cells to repair a critical size defect of the rat mandibular body.Prospective, randomized controlled trial.Thirty-seven male Fischer rats.Critical size defects 4 mm in diameter were created in the left mandibular bodies of the rats. The defects were filled with a bone marrow cell suspension (group 1), a synthetic bone matrix consisting of bovine collagen and calcium hydroxyapatite cement (group 2), the matrix and marrow cells (group 3), the matrix with 100 micro g of bone growth factor mixture (group 4), or the matrix with bone growth factor mixture and marrow cells (group 5). Animals were killed after 8 weeks, and the nondemineralized specimens were processed histologically. Specimens from group 1 were not processed because there was no grossly appreciable bone regeneration. Stereologic techniques were used to determine and compare the volume fractions and volume estimates of mature bone, new bone, osteoid, marrow, remaining cement, and fibrous tissue in each defect.Volumes of mature bone, new bone, and remaining cement did not differ significantly among the groups (P =.30 for mature bone, P =.17 for new bone, and P =.34 for cement). However, group 4 and 5 specimens contained significantly more osteoid and larger marrow spaces than did the group 2 and 3 specimens (P.001 for both). The specimens in groups 2 and 3 contained significantly more fibrous tissue ingrowth than did those in groups 4 and 5 (P.001).The synthetic bone substitute containing bone growth factor mixture was effective in stimulating new bone and osteoid development in the rat mandibular model.

Hydroxyapatite- (HA-)based ceramics have been evaluated for a variety of applications in spinal surgery, utilizing in vivo animal models and human clinical series. In vivo animal studies have shown efficacy for these materials as a bone graft substitute in interbody fusions and as a bone graft extender or bioactive osteoinductive material carrier in posterolateral lumbar fusions. Clinically, HA ceramic has been shown to be effective as a bone graft extender in posterior spinal fusion surgery for childhood scoliosis, and as a structural bone graft substitute in anterior cervical spine fusions. As an osteoconductive material, it appears to function best as a bone graft extender or carrier for an osteoinductive bone growth factor rather than as a stand-alone bone graft substitute in nonstructural clinical applications. Injectable HA ceramics also hold promise as biocompatible and bioresorbable materials for use in spinal screw fixation strength augmentation and in minimally invasive vertebral body strength augmentation either following fracture or prophylactically in osteoporotic vertebrae.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10–30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Study Design. The effect on spinal fusion of an osteoinductive bone protein extract in the presence of a known inhibitor of spinal fusion (systemic nicotine) was studied prospectively in an animal model of posterolateral lumbar fusion. Objectives. To evaluate the ability of a bovine-derived osteoinductive bone protein extract to overcome the inhibitory effect of nicotine in a rabbit spine fusion model. Summary of Background Data. Multiple studies have demonstrated the ability of a variety of osteoinductive growth factors to serve as a bone graft substitute for lumbar spinal fusion under "normal" healing conditions. Methods. Forty-eight adult female New Zealand white rabbits underwent spine arthrodesis at L5-L6 while receiving systemic nicotine through a subcutaneous miniosmotic pump. Arthrodesis was performed using one of the following three graft materials: 1) autogenous iliac crest, 2) osteoinductive bone protein delivered in an allogeneic demineralized bone matrix/collagen carrier, or 3) osteoinductive bone protein delivered with autogenous iliac crest. Fusions were assessed by blinded manual palpation, radiography, and biomechanical testing. Results. Of the 44 rabbits manually tested by blinded observers, all 14 in the osteoinductive bone protein plus autogenous iliac crest bone group had solid fusions (14 of 14), whereas the fusion rate was less in the osteoinductive bone protein plus demineralized bone matrix group (nine of 14, 64%; P = 0.02), and there were no fusions in the autogenous iliac crest only group (0 of 16, 0%; P = 0.000001). The use of osteoinductive bone protein with autogenous bone produced stronger and stiffer fusions compared with those using autogenous bone alone or osteoinductive bone protein with allograft bone. Conclusions. Cigarette smoking and nicotine are inhibitory factors in the healing of fractures and spine fusions. This study shows that the inhibitory effect of nicotine can be overcome with an osteoinductive bone growth factor in an animal model.

Bone growth and remodeling are controlled by local and systemic growth factors. The first local bone growth factor purified to homogeneity was transforming growth factor type beta (TGF-beta). On skeletal cells, TGF-beta has multiple effects mediated through at least three distinct cell surface receptors. More recent evidence demonstrated hormone and growth factor dependent alterations in TGF-beta receptor expression on osteoblasts in vitro. Indeed, certain biological responses appear to depend on the proportional expression of the type I TGF-beta receptor. Studies defining the type I TGF-beta receptor gene promoter then revealed that it contained several binding sequences for a nuclear factor that varies in parallel with expression of the osteoblast phenotype. New observations linking these events appear to enhance our understanding of this pivotal growth factor during osteogenesis and systemic bone disease.

In reviewing the scientific literature of hard-tissue repair/generation, it can be concluded that it is worthwhile to evaluate the possibility of obtaining synergistic effects by combining bioresorbable scaffolds with other factors stimulatory to osteogenesis. Theoretically, this can occur at different principal levels. Cytokines, including growth factors such as BMP, the IGFs, TGF-b, PDGF, may be stimulatory to the differentiation of cells of the osteoblastic lineage, thus having the potential to promote an increased recruitment of osteogenic cells. The IGFs, TGF-b, and PDGF are known to stimulate the synthetic capacity of mature osteoblasts. In addition, the FGFs are angiogenic and may thus improve nutrition for a healing bone defect by an early establishment of the vascular bed. At present, the field of growth-stimulatory factors is strongly expanding. Even though the effects on bone of such factors are far from being completely elucidated, it may be expected that some of them will be of clinical relevance in the future. Future developments of de novo biodegradable and bioresorbable carrier and matrix materials treated with growth factors should have the objectives: biointeractive and biomi-metic devices/implants endowed with cell or cell-based signals; a synthetic extracellular matrix for enhanced cell interaction, cell polarization, or remodeling; and a temporal and/or spatial delivery of bioactive agents over short and long time-periods.