Growth differentiation factor-11 supplementation improves survival and promotes recovery after ischemic stroke in aged mice.

Growth differentiation factor (GDF) 11 levels decline with aging. The age-related loss of GDF 11 has been implicated in the pathogenesis of a variety of age-related diseases. GDF11 supplementation reversed cardiac hypertrophy, bone loss, and pulmonary dysfunction in old mice, suggesting that GDF11 has a rejuvenating effect. Less is known about the potential of GDF11 to improve recovery after an acute injury, such as stroke, in aged mice. GDF11/8 levels were assessed in young and aged male mice and in postmortem human brain samples. Aged mice were subjected to a transient middle cerebral artery occlusion (MCAo). Five days after MCAo, mice received and bromodeoxyuridine / 5-Bromo-2'-deoxyuridine (BrdU) and either recombinant GDF11 or vehicle for five days and were assessed for recovery for one month following stroke. MRI was used to determine cerebrospinal fluid (CSF) volume, corpus callosum (CC) area, and brain atrophy at 30 days post-stroke. Immunohistochemistry was used to assess gliosis, neurogenesis, angiogenesis and synaptic density. Lower GDF11/8 levels were found with age in both mice and humans (p<0.05). GDF11 supplementation reduced mortality and improved sensorimotor deficits after stroke. Treatment also reduced brain atrophy and gliosis, increased angiogenesis, improved white matter integrity, and reduced inflammation after stroke. GDF11 may have a role in brain repair after ischemic injury.

The Elution Kinetics of BMP-2, BMP-4, and BMP-7 From a Commercial Human Demineralized Bone Matrix Putty.

Bone morphogenetic proteins (BMPs) are associated with bone extracellular matrix and impart osteoinductive properties to demineralized bone matrix (DBM) grafts. The first step of the osteoinductive process is BMP release from DBM in situ; however, this has not been characterized for human DBM. The authors investigated the release of BMPs 2, 4, and 7 from a clinical human DBM putty (Bonus II DBM, Biomet Inc, Warsaw, IN). The DBM was placed in Sorensen buffer and the BMP concentrations in the Sorensen buffer and guanidine extracts of the DBM were measured concurrently by enzymelinked immunosorbant assay for up to 7 days. The baseline DBM concentrations were BMP-2: 28.1 ±â€Š1.3 ng/g DBM, BMP-4: 0.577 ±â€Š0.056 ng/g DBM, and BMP-7: 92.9 ±â€Š7.5 ng/g DBM. Relative to baseline, the proportions released by 7 days were 11.1%, 3.9%, and 29.3%, respectively. The early (0-8 hour) and late (8-168 hours) elution rates were BMP-2: 0.16 ±â€Š0.24 and 0.0089 ±â€Š0.012 ng/(g DBM hr), and BMP-7: 1.29 ±â€Š2.1 and 0.086 ±â€Š0.039 ng/(g DBM hr), respectively. Little BMP-4 elution occurred over the first 24 hours, with the rate for the remaining interval being 0.00014 ±â€Š0.00021 ng/(g DBM hr). The apparent DBM BMP profiles were counterintuitive in that the concentrations increased from baseline for some, or all, of the 7 days instead of monotonically decreasing. Similar behavior has previously been reported in bovine studies. This provides further evidence that BMPs are associated with at least 2 compartments in DBM differing by their affinity for BMPs and that guanidine extraction of BMPs is not 100% efficient.

Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform.

Decisions about efficacy and safety of therapeutic proteins (TP) designed to target soluble ligands are made in part by their ex vivo quantification. Ligand binding assays (LBAs) are critical tools in measuring serum TP levels in pharmacokinetic, toxicokinetic, and pharmacodynamic studies. This study evaluated the impact of reagent antibody affinities, assay incubation times, and analytical platform on free or total TP quantitation. An ELISA-based LBA that measures monoclonal anti-sclerostin antibody (TPx) was used as the model system. To determine whether the method measures free or total TPx, the effects of K on, K off, and K D were determined. An 8:1 molar ratio of sclerostin (Scl) to TPx compared to a 1:1 molar ratio produced by rabbit polyclonal antibodies to TPx was required to achieve IC50, a measure of TPx interference effectiveness, making it unclear whether the ELISA truly measured free TPx. Kinetic analysis revealed that Scl had a rapid dissociation rate (K off) from TPx and that capture and detection antibodies had significantly higher binding affinities (K D) to TPx. These kinetic limitations along with long ELISA incubation times lead to the higher molar ratios (8:1) required for achieving 50% inhibition of TPx. However, a microfluidic platform with the same reagent pairs required shorter incubations to achieve a lower Scl IC50 molar ratio (1:1). The findings from this study provide the bioanalytical community with a deeper understanding of how reagent and platform selection for LBAs can affect what a particular method measures, either free or total TP concentrations.

Local rhBMP-12 on an Absorbable Collagen Sponge as an Adjuvant Therapy for Rotator Cuff Repair - A Phase 1, Randomized, Standard of Care Control, Multicenter Study: Safety and Feasibility.

BACKGROUND: Recombinant human bone morphogenetic protein-12 (rhBMP-12) has been shown to induce tendon and ligament formation in rats and to improve tendon healing; however, the safety and feasibility of implanting rhBMP-12/absorbable collagen sponge (ACS) in humans are not known. PURPOSE: To investigate the safety and feasibility of rhBMP-12 on an ACS as an adjuvant therapy in open rotator cuff repair. STUDY DESIGN: Randomized controlled trial; Level of evidence, 2. METHODS: This study consisted of 20 patients with full-thickness rotator cuff tears. Patients were randomized either to standard of care (SOC) treatment (open rotator cuff repair) or to receive 0.015 mg/mL rhBMP-12/ACS and SOC treatment during their open rotator cuff repair (rhBMP-12/ACS group) at a rate of 1/4 SOC/rhBMP-12/ACS. The feasibility of implanting the product and the safety of the product were evaluated during the 1-year follow-up period. The evaluation involved up to 10 postoperative visits, which included physical examinations, radiographs, computed tomography (CT) scans, magnetic resonance imaging (MRI) scans with an emphasis on heterotopic ossification (HO), pharmacokinetics, immunogenicity, laboratory evaluations, and local and systemic adverse events at specified time points. RESULTS: Small amounts of HO were seen on follow-up CT scans in 10 of 16 patients in the rhBMP-12/ACS group and in 2 of 3 patients in the SOC group. HO did not increase at 26 weeks and was not associated with any adverse events or unsatisfactory clinical outcomes. Pharmacokinetics demonstrated that circulating levels of rhBMP-12 were not detectable after administration. Five of 16 patients showed a postoperative immunogenic response but did not show any correlating adverse events. Complete healing of the rotator cuff was observed in 14 of 16 patients; 2 of 16 imaging results could not be analyzed because of artifacts in the rhBMP-12 group on MRI scans. In the SOC group, 1 of 4 patients showed a retear at 12 weeks after surgery. CONCLUSION: The use of rhBMP-12/ACS has been shown to be feasible and safe in a concentration of 0.015 mg/mL when used in open rotator cuff repair. Higher dose concentrations of rhBMP-12 should be evaluated in the future to evaluate their safety and potential to increase rotator cuff healing after open surgical repair.

Use of bone morphogenetic proteins in spinal fusion surgery for older adults with lumbar stenosis: trends, complications, repeat surgery, and charges.

STUDY DESIGN: Retrospective cohort study of Medicare claims. OBJECTIVE: Examine trends and patterns in the use of bone morphogenetic proteins (BMP) in surgery for lumbar stenosis; compare complications, reoperation rates, and charges for patients undergoing lumbar fusion with and without BMP. SUMMARY OF BACKGROUND DATA: Small, randomized trials have demonstrated higher rates of solid fusion with BMP than with allograft bone alone, with few complications and, in some studies, reduced rates of revision surgery. However, complication and reoperation rates from large population-based cohorts in routine care are unavailable. METHODS: We identified patients with a primary diagnosis of lumbar stenosis who had fusion surgery in 2003 or 2004 (n = 16,822). We identified factors associated with BMP use: major medical complications during the index hospitalization, rates of rehospitalization within 30 days, and rates of reoperation within 4 years of follow-up (through 2008). RESULTS: Use of BMP increased rapidly, from 5.5% of fusion cases in 2003 to 28.1% of fusion cases in 2008. BMP use was greater among patients with previous surgery and among those having complex fusion procedures (combined anterior and posterior approach, or greater than 2 disc levels). Major medical complications, wound complications, and 30-day rehospitalization rates were nearly identical with or without BMP. Reoperation rates were also very similar, even after stratifying by previous surgery or surgical complexity, and after adjusting for demographic and clinical features. On average, adjusted hospital charges for operations involving BMP were about $15,000 more than hospital charges for fusions without BMP, though reimbursement under Medicare's Diagnosis-Related Group system averaged only about $850 more. Significantly fewer patients receiving BMP were discharged to a skilled nursing facility (15.9% vs. 19.0%, P < 0.001). CONCLUSION: In this older population having fusion surgery for lumbar stenosis, uptake of BMP was rapid, and greatest among patients with prior surgery or having complex fusion procedures. BMP appeared safe in the perioperative period, with no increase in major medical complications. Use of BMP was associated with greater hospital charges but fewer nursing home discharges, and was not associated with reduced likelihood of reoperation.

Reconstruction of the alveolar cleft: can growth factor-aided tissue engineering replace autologous bone grafting? A literature review and systematic review of results obtained with bone morphogenetic protein-2.

The alveolar cleft in patients with clefts of lip, alveolus and palate (CLAP) is usually reconstructed with an autologous bone graft. Harvesting of autologous bone grafts is associated with more or less donor site morbidity. Donor site morbidity could be eliminated if bone is fabricated by growth factor-aided tissue engineering. The objective of this review was to provide an oversight on the current state of the art in growth factor-aided tissue engineering with regard to reconstruction of the alveolar cleft in CLAP. Medline, Embase and Central databases were searched for articles on bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7, transforming growth factor beta, platelet-derived growth factor, insulin-like growth factor, fibroblast growth factor, vascular endothelial growth factor and platelet-rich plasma for the reconstruction of the alveolar cleft in CLAP. Two-hundred ninety-one unique search results were found. Three articles met our selection criteria. These three selected articles compared BMP-2-aided bone tissue engineering with iliac crest bone grafting by clinical and radiographic examinations. Bone quantity appeared comparable between the two methods in patients treated during the stage of mixed dentition, whereas bone quantity appeared superior in the BMP-2 group in skeletally mature patients. Favourable results with BMP-2-aided bone tissue engineering have been reported for the reconstruction of the alveolar cleft in CLAP. More studies are necessary to assess the quality of bone. Advantages are shortening of the operation time, absence of donor site morbidity, shorter hospital stay and reduction of overall cost.

Formulations and delivery vehicles for bone morphogenetic proteins: latest advances and future directions.

Growth factors are essential components of the diamond concept model. The bone morphogenetic proteins (BMPs) are the most potent and promising growth factors and their clinical efficacy is well demonstrated for specific indications. Application of BMPs involves a carrier material to enhance local residual time and pharmacokinetics. On the other hand carrier materials, collagen at this point, also limit the use of BMPs, for example in minimally invasive application methods. In this overview, the pharmacokinetics of BMPs, and various carrier materials (collagen, synthetic polymers, calcium phosphates, hyaluronic acid, CMC, and sodium acetate) are discussed. No other carrier material than collagen has been proven effective in clinical studies. Other formulations are needed to improve the residual time and handling.

Bone morphogenetic proteins and tissue engineering: future directions.

As long as bone repair and regeneration is considered as a complex clinical condition, the administration of more than one factor involved in fracture healing might be necessary. The effectiveness or not of bone morphogenetic proteins (BMPs) in association with other growth factors and with mesenchymal stem cells in bone regeneration for fracture healing and bone allograft integration is of great interest to the scientific community. In this study we point out possible future developments in BMPs, concerning research and clinical applications.

BMP-7 protects against progression of cartilage degeneration after impact injury.

In vivo studies were used to characterize a model of cartilage injury leading to osteoarthritis progression in the medial femorotibial joint of sheep. In three subsequent studies, bilateral impact injuries were created and one joint received intraarticular injections of 340 microg of rhBMP-7 protein in a collagen particle carrier while the contralateral knee received the vehicle alone. Sheep were allocated to three groups that received intraarticular injections on day 0 (group A), 21 (group B), or 90 (group C) after experimental knee injury. In each group the, joints were evaluated for signs of osteoarthritis progression 90 days after the last treatment using India ink stained area, OARSI histological scoring, cartilage sGAG content, immunostaining for apoptosis (TUNEL), caspase-3, collagen degradation (Col 2 3/4C short collagen epitope), and the endogenous (pro-) form of BMP-7 protein. Knee joints that received rhBMP-7 immediately after injury had small focal lesions at the injury site that did not progress into the surrounding cartilage. Joints that received BMP-7 3 weeks after injury were improved and had limited progression compared to controls, but joints that received the protein 12 weeks after injury had no statistically significant improvement. These studies suggest that BMP-7 may be chondroprotective after traumatic injury in patients if it is administered within 3 to 4 weeks of the index injury. The mechanism of protection after sublethal injury appeared to be an increased survival of chondrocytes that are able to participate in the repair process.

Non-invasive screening method for simultaneous evaluation of in vivo growth factor release profiles from multiple ectopic bone tissue engineering implants.

The purpose of this study was to develop and validate a screening method based on scintillation probes for the simultaneous evaluation of in vivo growth factor release profiles of multiple implants in the same animal. First, we characterized the scintillation probes in a series of in vitro experiments to optimize the accuracy of the measurement setup. The scintillation probes were found to have a strong geometric dependence and experience saturation effects at high activities. In vitro simulation of 4 subcutaneous limb implants in a rat showed minimal interference of surrounding implants on local measurements at close to parallel positioning of the probes. These characteristics were taken into consideration for the design of the probe setup and in vivo experiment. The measurement setup was then validated in a rat subcutaneous implantation model using 4 different sustained release carriers loaded with (125)I-BMP-2 per animal. The implants were removed after 42 or 84 days of implantation, for comparison of the non-invasive method to ex vivo radioisotope counting. The non-invasive method demonstrated a good correlation with the ex vivo counting method at both time-points of all 4 carriers. Overall, this study showed that scintillation probes could be successfully used for paired measurement of 4 release profiles with minimal interference of the surrounding implants, and may find use as non-invasive screening tools for various drug delivery applications.

Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering.

In this study, we investigated the in vitro and in vivo biological activities of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated into (1) a gelatin hydrogel, (2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, (3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and (4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with (125)I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivities of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (Implant 3, p<0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than that in the gelatin and microsphere/gelatin hydrogels (p<0.001), however, there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.

125I-labeled OP-1 is locally retained in a rabbit lumbar fusion model.

Osteogenic protein-1 is evolving as a potential bone graft alternative. Surgical site retention is important to maximize local osteoinduction and to limit peripheral effects. An established rabbit lumbar posterolateral fusion model was used to evaluate the systemic distribution and pharmacokinetics of locally applied osteogenic protein-1 delivered on a collagen carrier. L5-L6 intertransverse process fusions were performed on 27 New Zealand White rabbits. Radiolabeled (125)I-osteogenic protein-1 collagen putty was implanted. At intervals, whole blood, plasma, and excreta were analyzed for radioactivity with liquid scintillation counting. Surgical site and tissue radioactivity also were assessed by quantitative whole-body autoradioluminography of animals euthanized at times ranging from 6 hours to 35 days. Animals remaining at the final time were assessed for fusion with manual palpation, radiography, and histology. Limited distribution of radioactivity was observed in the blood, plasma, and tissues apart from at the surgical site and in the urinary bladder and thyroid. The mean residence time for osteogenic protein-1 collagen putty was 10.4 +/- 2.7 days. These excretion profiles and kinetic properties are similar to those described for recombinant human bone morphogenetic protein-2 in the rabbit model (mean residence times of 7.6 days and 10.2 days with different carriers).

Preparation and property of a novel bone graft composite consisting of rhBMP-2 loaded PLGA microspheres and calcium phosphate cement.

Calcium phosphate cement (CPC) is a highly promising bone substitute and an excellent carrier for delivering growth factors. Yet, the lack of macro-porosity and osteoinductive ability, limit its use. This study is aimed at developing a novel biodegradable biomaterial for bone repair with both highly osteoconductive and osteoinductive properties. RhBMP-2 loaded PLGA microspheres were incorporated into rhBMP-2/CPC for macropores for bone ingrowth. The compressive strength, crystallinity, microscopic structure, and bioactivity of the composites were investigated. The results showed that with the incorporation of rhBMP-2 loaded PLGA microspheres, the compressive strength was decreased from (29.48+/-6.42) MPa to (8.26+/-3.58) MPa. X-ray diffraction revealed that the crystallinity pattern of HA formed by CPC had no significant change. Inside the composite, the microspheres distributed homogeneously and contacted intimately with the HA matrix, as observed by scanning electron microscopy (SEM). When the PLGA microspheres dissolved after having been emerged in PBS for 56 days, macropores were created within the CPC. The rhBMP-2/PLGA/CPC composite, showing a 4.9% initial release of rhBMP-2 in 24 h, followed by a prolonged release for 28 days, should have a greater amount of rhBMP-2 released compared to the CPC delivery system. When rabbit marrow stromal cells were cocultured with the composite, the alkaline phosphatase (ALP) and osteocalcin (OC) showed a dose response to the rhBMP-2 released from the composite, indicating that the activity of rhBMP-2 was retained. This study shows that the new composite reveals more rhBMP-2 release and osteogenic activity. This novel BMP/PLGA/CPC composite could be a promising synthetic bone graft in craniofacial and orthopedic repairs.

Understanding adsorption-desorption dynamics of BMP-2 on hydroxyapatite (001) surface.

The interaction between protein molecules and the hydroxyapatite (HAP) crystal is an important research topic in many fields. However, the nature of their noncovalent bonding is still not clear at the atomic level. In this work, molecular dynamics simulation, steered molecular dynamics simulation, and quantum chemistry calculations were used to study the adsorption-desorption dynamics of BMP-2 on HAP (001) surface. The results suggest that there are three types of functional groups through which BMP-2 can interact with HAP crystallite, and they are -OH, -NH(2), and -COO(-). Based on the different orientations of protein, each might interact with HAP crystallite individually, or, two or three of them can work cooperatively. Concerning the mechanisms of interaction, it is found that the water-bridged H-bond plays an important role, which is the main force for groups without net charges. If there were more than one set of adsorption groups for a certain orientation of protein, the adsorption-desorption process would likely be stepwise. On the contrary, if there were only one set, there would be only the key-adsorption period. The results of density functional theory calculations confirm the actual existence of this type of water-bridged H-bond. Furthermore, it is also found that the CHARMM27 force field could provide correct structural information qualitatively, although the data are slightly different from those obtained by UB3LYP/6-31G* method.

Pharmacokinetics and biological effect of the recombinant human bone morphogenetic protein-7.

BACKGROUND: During Bone Morphogenetic Protein (BMP) induced osteoblastogenesis and bone formation, strong vascularization is observed at the transition of preosteoblasts to mature osteoblasts. The cellular and molecular mechanisms that determine autonomous hematopoietic competence in the BMP induced osteoblastogenesis and angiogenesis have not been characterized yet. AIMS: In the present study we investigated, whether rhBMP-7 in collagen as carrier (ACS) stimulates osteoblastogenesis have effect on the physiologic autonomous hematopoetic system and clinical condition on animal model. MATERIAL AND METHOD: Study was' performed in the Clinic for Maxillofacial Surgery University Clinic Center Sarajevo from 2003 to 2004. Mandible hemiresection non-critical size created defect were treated with rhBMP7/ACS on 7 rabbits, and on other group of 7 defects were treated with autologues bone graft iliac crest. Biochemical and hematologist tests were performed measurement Alkaline Phosphates enzyme (ALP) activity and Erythrocytes (Er), Leukocytes (Le), Hematocrit(HCR) and Hemoglobin(Hg) count extracted from the periphery blood preoperative, 5, 14, 21, 30 day. RESULT: ALP activity showed strong osteoinductive effect rhBMP-7 determinate with day 21 significance increased activity and on bone graft sites day 30. All hematologist values were in physiologic range on both groups. CONCLUSION: Strong osteoinductive response rhBMP/ACS construct determinate by the higher value of the ALP enzyme didn't affect hematopoetic competence determinate by the Er. Le, HCR and Hg. counts parameters. Result indicated that BMP-inducedangiogenesis may thus be the result of BMP-induced secretion some angiogenetic factor locally from osteoblasts named Vascular Endothelial Growth Factor (VEGF).

Estudio densitométrico del tejido óseo neoformado por el uso de rhBMP-2 en mandíbulas de ratas Wistar / Optical densitometry study of the newly formed bone using rhBMP-2 in Wistar rat mandibles

The purpose of this study was to evaluate the efficacy of monoolein gel as a carrier for rhBMP-2 in the healing bone process of critical bone defects created in Wistar rats mandibles using digitalized radiographic method to analyze this process. In the group 1, the rhBMP-2 was dissolved in aqueous solution and in the group 2, the rhBMP-2 was combined with monoolein gel as a carrier. The results showed that in both of groups it was found efficient bone repair, with a great optical density in the group that the rhBMP-2 was combined with the monoolein gel, but without statistical difference between them.

El objetivo de este estudio fue evaluar la eficiencia del gel de monoolein como portador de la rhBMP-2 en el proceso de recuperación de la osificación en defectos osteos creados en mandíbulas de ratos Wistar, por medio de radiografias digitalizadas como método de análisis. En el grupo 1, la rhBMP-2 fue disuelta en solución acuosa y en el grupo 2, la rhBMP-2 fue combinada al gel de monoolein como portador. Los resultados mostraron que los dos grupos presentaron un eficiente reparo osteo, con mayor densidad óptica en el grupo en que la rhBMP-2 fue combinada con el gel de monoolein, pero sin diferencia estadística significante entre ellos.

Homogeneous osteogenesis and bone regeneration by demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2.

Considerable research has been focused on the development of bone morphogenetic protein-2 (BMP-2) delivery system for homologous and efficient bone regeneration. The aim of the present study was to develop a collagen-based targeting bone repair system. A collagen-binding domain (CBD) was added to the N-terminal of native BMP-2 to allow it bind to collagen specifically. We showed that the collagen-binding bone morphogenetic protein-2 (named bone morphogenetic protein2-h, BMP2-h) had maintained the full biological activity as compared to rhBMP2 lacking the CBD. In vitro functional study also demonstrated that collagen matrix could maintain higher bioactivity of BMP2-h than native BMP-2. When demineralized bone matrix (DBM) impregnated with BMP2-h was implanted subcutaneously in rats, homogeneous bone formation was observed. Moreover, in a rabbit mandible defect model, surgical implantation of collagen matrix loaded with BMP2-h exhibited remarkable osteoinductive properties and excellent homogeneous bone formation. Our studies suggested that this novel collagen-based BMP-2 targeting bone repair system induced better bone formation not only in quantity but also in quality. Similar approaches may also be used for the repair of other tissue injuries.

Zonal release of proteins within tissue engineering scaffolds.

The manufacture of a scaffold for tissue engineering applications that can control the location and timing of growth factor release is described. The scaffold is formed by the sintering of poly(DL-lactic acid) (P(DL)LA) microparticles, plasticized with poly(ethylene glycol) (PEG), although the method can be used for many other polymer types. The microparticles were loaded with model proteins, trypsin and horseradish peroxidase (HRP), or recombinant human bone morphogenetic protein-2 (rhBMP-2). Entrapment efficiencies above 75% were achieved using a solid-in-oil-in-water system. Controlled release of active protein was achieved for at least 30 days. Microparticles were built into protein-loaded or protein-free layers and release of the protein was restricted to zones within the scaffold. Cell response to rhBMP-2 was tuneable by changing the dose of the rhBMP-2 released by varying the ratio of protein-loaded and protein-free microparticles within scaffolds. Zonal activity of rhBMP-2 on C2C12 cells was demonstrated. The scaffolds may find utility in applications where gradients of growth factors within 3D templates are required or where zonation of tissue growth is required.

Targeted delivery system for juxtacrine signaling growth factor based on rhBMP-2-mediated carrier-protein conjugation.

We propose a model of artificial juxtacrine signaling for the controlled release of recombinant human bone morphogenetic protein-2 (rhBMP-2) suitable for guided bone regeneration. A porous three-dimensional scaffold of poly-(lactide-co-glycolide) was fabricated by means of gel molding and particulate leaching. Collagen immobilization onto the scaffold surface was produced by performing photo-induced graft polymerization of acrylic acid, and rhBMP-2 was tethered to the collagenous surface by covalent conjugation. On pharmacokinetic analysis, in vitro enzyme-linked immunosorbent and alkaline phosphatase assays revealed sustained, slow release of rhBMP-2 over 28 days, with a cumulative release of one third of the initial load diffusing out of the scaffold. Conjugation of rhBMP-2 inhibited the free lateral diffusion and internalization of the activated complex of rhBMP-2 and the bone morphogenetic protein receptor. Osteoprogenitor cells were used as bone precursors to determine the expression of biosignaling growth factor in regulating cell proliferation and differentiation. To identify the phenotype of cells seeded on the rhBMP-2-conjugated scaffold, cellular activity was evaluated with scanning electron microscopy and with viability, histological, and immunohistochemical testing. The rhBMP-2-conjugated scaffold prolonged stimulation of intracellular signal proteins in cells. Enhancement of cell growth and differentiation was considered a consequence of juxtacrine signaling transduction. Animal studies of rhBMP-2-containing filling implants showed evidence of resorption and de novo bone formation. The present study revealed the potential of biomimetic constructs with co-immobilized adhesion and growth factors to induce osteoinduction and osteogenesis. Such constructs may be useful as synthetic bone-graft materials in orthopaedic tissue engineering.

Controlling bone morphogenetic protein diffusion and bone morphogenetic protein-stimulated bone growth using fibrin glue.

STUDY DESIGN: An in vitro and in vivo study. OBJECTIVE: To evaluate the ability of fibrin glue to limit diffusion of recombinant human bone morphogenetic protein (rhBMP)-2 and its ability to protect spinal nerves from rhBMP-2 stimulated bone growth. SUMMARY OF BACKGROUND DATA: Studies have shown bone morphogenetic protein (rhBMP-2) stimulated bone growth can encroach on the spinal canal and nerves, causing neural compression. More recently, rhBMP-2 use in the cervical spine has been associated with life-threatening swelling. Fibrin glue has been used as a biologic carrier but has not been evaluated for its ability to limit rhBMP-2. METHODS: In phase 1 of the study, rhBMP-2 soaked absorbable collagen sponges (ACS) were encapsulated in fibrin glue and immediately incubated in physiologic lactated ringers solution at 38 degrees C. Samples of solution were tested for rhBMP-2 concentration. In phase 2 of the study, rats were surgically treated with laminectomy and placement of rhBMP-2/ACS versus laminectomy and placement of fibrin glue before placement of rhBMP-2/ACS. After 8 weeks, animals were euthanized and imaged using micro-computerized tomography. RESULTS: The diffusion study showed a significant limitation in rhBMP-2 diffusion when encapsulated in fibrin glue. The laminectomy study revealed blockage of bone formation by fibrin glue and protection of the spinal canal. CONCLUSIONS: Fibrin glue can limit the diffusion of rhBMP-2, and, thus, it can be used to help protect the spinal canal and nerve roots from rhBMP-2 stimulated bone growth.