Spatial Recombinant Human Bone Morphogenetic Protein 2 Delivery from Hydroxyapatite Scaffolds Sustains Bone Regeneration in Rabbit Radius.

Regenerating large bone defects requires a multifaceted approach combining optimal scaffold designs with appropriate growth factor delivery. Supraphysiological doses of recombinant human bone morphogenetic protein 2 (rhBMP2), typically used for the regeneration of large bone defects clinically in conjunction with an acellular collagen sponge (ACS), have resulted in many complications. In this study, we develop a hydroxyapatite/collagen I (HA/Col) scaffold to improve the mechanical properties of the HA scaffolds, while maintaining open connected porosity. Varying rhBMP2 dosages were then delivered from a collagenous periosteal membrane and paired with HA or HA/Col scaffolds to treat critical-sized (15 mm) diaphyseal radial defect in New Zealand white rabbits. The groups examined were ACS +76 µg rhBMP2 (clinically used INFUSE dosage), HA +76 µg rhBMP2, HA +15 µg rhBMP2, HA/Col +15 µg rhBMP2, and HA/Col +15 µg rhBMP2 + bone marrow-derived stromal cells (bMSCs). After 8 weeks of implantation, all regenerated bones were evaluated using microcomputed tomography, histology, histomorphometry, and torsional testing. It was observed that the bone volume regenerated in the HA/Col +15 µg rhBMP2 group was significantly higher than that in the groups with 76 µg rhBMP2. The same scaffold and growth factor combination resulted in the highest bone mineral density of the regenerated bone, and the most bone apposition on the scaffold surface. Both the HA and HA/Col scaffolds paired with 15 µg rhBMP2 had sustained ingrowth of the mineralization front after 2 weeks compared to the groups with 76 µg rhBMP2, which had far greater mineralization in the first 2 weeks after implantation. Complete bridging of the defect site and no significant difference in torsional strength, stiffness, or angle at failure were observed across all groups. No benefit of additional bMSC seeding was observed on any of the quantified metrics, while bone-implant apposition was reduced in the cell-seeded group. This study demonstrated that the controlled spatial delivery of rhBMP2 at the periosteum at significantly lower doses can be used as a strategy to improve bone regeneration around space maintaining scaffolds. Tweet Inside-out or outside-in: growth factors delivered from the outside of porous mineral-collagen scaffolds, maintain strength and regrow bone better in a rabbit study. Twitter handle for senior author (@Guda_Lab) and sponsoring institution (@UTSA) Impact Statement This study provides insights on bone regeneration in the presence of spatially controlled delivery of recombinant human bone morphogenetic protein 2 (rhBMP2) from porous hydroxyapatite scaffolds coated with collagen I films. Using critical-sized defects created in the radial diaphysis of skeletally mature New Zealand White rabbits, microcomputed tomography and histomorphometry indicated significantly higher bone regeneration, bone mineral density, and bone-implant contact, as well as sustained regeneration over longer durations with lower dosage of rhBMP2 delivered periosteally.

Regeneration enhanced in critical-sized bone defects using bone-specific extracellular matrix protein.

Extracellular matrix (ECM) products have the potential to improve cellular attachment and promote tissue-specific development by mimicking the native cellular niche. In this study, the therapeutic efficacy of an ECM substratum produced by bone marrow stem cells (BM-MSCs) to promote bone regeneration in vitro and in vivo were evaluated. Fluorescence-activated cell sorting analysis and phenotypic expression were employed to characterize the in vitro BM-MSC response to bone marrow specific ECM (BM-ECM). BM-ECM encouraged cell proliferation and stemness maintenance. The efficacy of BM-ECM as an adjuvant in promoting bone regeneration was evaluated in an orthotopic, segmental critical-sized bone defect in the rat femur over 8 weeks. The groups evaluated were either untreated (negative control); packed with calcium phosphate granules or granules+BM-ECM free protein and stabilized by collagenous membrane. Bone regeneration in vivo was analyzed using microcomputed tomography and histology. in vivo results demonstrated improvements in mineralization, osteogenesis, and tissue infiltration (114 ± 15% increase) in the BM-ECM complex group from 4 to 8 weeks compared to mineral granules only (45 ± 21% increase). Histological observations suggested direct apposition of early bone after 4 weeks and mineral consolidation after 8 weeks implantation for the group supplemented with BM-ECM. Significant osteoid formation and greater functional bone formation (polar moment of inertia was 71 ± 0.2 mm4 with BM-ECM supplementation compared to 48 ± 0.2 mm4 in untreated defects) validated in vivo indicated support of osteoconductivity and increased defect site cellularity. In conclusion, these results suggest that BM-ECM free protein is potentially a therapeutic supplement for stemness maintenance and sustaining osteogenesis.

In vivo hydroxyapatite scaffold performance in infected bone defects.

Critically sized bone defects are often compounded by infectious complications. The standard of care consists of bone autografts with systemic antibiotics. These injuries and treatments lead to donor site morbidity, antibiotic resistant strains of bacteria, and often end stage amputation. This study proposes an alternative to the autograft using a porous, hydroxyapatite (HA) scaffold evaluated with and without infection and antibiotics. Twenty-four New Zealand white rabbits received either our HA scaffold or a pulverized autograft (PBA) within a surgically created critical-sized defect in the femur. The two grafts were evaluated in either septic or aseptic defects and with or without antibiotic treatment. The HA scaffolds were characterized with micro computed tomography. Post-euthanasia, micro computed tomography, histology, and white blood cells component analysis were completed. The HA had significantly greater (p < .001) mineralization to total volume than the PBA groups with 27.56% and 14.88%, respectively, and the septic HA groups were significantly greater than the aseptic groups both with and without antibiotics (p = .016). The bone quality denoted by bone mineral density was also significantly greater (p < .001) in the HA groups (67.01 ± 0.38 mgHA/cm3 ) than the PBA groups (64.66 ± 0.85 mgHA/cm3 ). The HA scaffold is a viable alternative to the bone autograft in defects with and without infection as shown by the quality and quantity of bone.

Bone Formation is Affected by Matrix Advanced Glycation End Products (AGEs) In Vivo.

Advanced glycation end products (AGEs) accumulate in bone extracellular matrix as people age. Although previous evidence shows that the accumulation of AGEs in bone matrix may impose significant effects on bone cells, the effect of matrix AGEs on bone formation in vivo is still poorly understood. To address this issue, this study used a unique rat model with autograft implant to investigate the in vivo response of bone formation to matrix AGEs. Fluorochrome biomarkers were sequentially injected into rats to label the dynamic bone formation in the presence of elevated levels of matrix AGEs. After sacrificing animals, dynamic histomorphometry was performed to determine mineral apposition rate (MAR), mineralized surface per bone surface (MS/BS), and bone formation rate (BFR). Finally, nanoindentation tests were performed to assess mechanical properties of newly formed bone tissues. The results showed that MAR, MS/BS, and BFR were significantly reduced in the vicinity of implant cores with high concentration of matrix AGEs, suggesting that bone formation activities by osteoblasts were suppressed in the presence of elevated matrix AGEs. In addition, MAR and BFR were found to be dependent on the surrounding environment of implant cores (i.e., cortical or trabecular tissues). Moreover, MS/BS and BFR were also dependent on how far the implant cores were away from the growth plate. These observations suggest that the effect of matrix AGEs on bone formation is dependent on the biological milieu around the implants. Finally, nanoindentation test results indicated that the indentation modulus and hardness of newly formed bone tissues were not affected by the presence of elevated matrix AGEs. In summary, high concentration of matrix AGEs may slow down the bone formation process in vivo, while imposing little effects on bone mineralization.

Pretensioning of Soft Tissue Grafts in Anterior Cruciate Ligament Reconstruction.

To determine which preconditioning and pretensioning techniques should be applied to soft tissue grafts during anterior cruciate ligament (ACL) reconstruction to avoid loss of tension after surgery, fresh semitendinosus and tibialis anterior tendons underwent tensile mechanical testing with 4 pretensioning and/or preconditioning techniques. A mechanical tester was used to collect the data. Group I (n=5) was given only an initial 80 N pull for tensioning, Group II (n=4) was given pretensioning and initial tensioning, Group III (n=5) was given cyclic tensioning and initial tensioning, and Group IV (n=5) was given a combination of the 3 techniques. Group I lost 50% of the initial tension at 30 minutes. The residual tension in Groups II, III, and IV was significantly higher than that in Group I after 1, 10, and 30 minutes (P<.001). Group IV consistently showed significantly higher residual tension than Groups II and III after 10 and 30 minutes (P<.05). All groups experienced elongation during testing: Group I (10.8 mm)

Age-Related Effects of Advanced Glycation End Products (Ages) in Bone Matrix on Osteoclastic Resorption.

Advanced glycation end products (AGEs) accumulate in bone extracellular matrix as people age. Previous studies have shown controversial results regarding the role of in situ AGEs accumulation in osteoclastic resorption. To address this issue, this study cultured human osteoclast cells directly on human cadaveric bone slices from different age groups (young and elderly) to warrant its relevance to in vivo conditions. The cell culture was terminated on the 3rd, 7th, and 10th day, respectively, to assess temporal changes in the number of differentiated osteoclasts, the number and size of osteoclastic resorption pits, the amount of bone resorbed, as well as the amount of matrix AGEs released in the medium by resorption. In addition, the in situ concentration of matrix AGEs at each resorption pit was also estimated based on its AGEs autofluorescent intensity. The results indicated that (1) osteoclastic resorption activities were significantly correlated with the donor age, showing larger but shallower resorption pits on the elderly bone substrates than on the younger ones; (2) osteoclast resorption activities were not significantly dependent on the in situ AGEs concentration in bone matrix, and (3) a correlation was observed between osteoclast activities and the concentration of AGEs released by the resorption. These results suggest that osteoclasts tend to migrate away from initial anchoring sites on elderly bone substrate during resorption compared to younger bone substrates. However, such behavior is not directly related to the in situ concentration of AGEs in bone matrix at the resorption sites.

Evaluation of Amniotic Multipotential Tissue Matrix to Augment Healing of Demineralized Bone Matrix in an Animal Calvarial Model.

Amniotic multipotential tissue matrix (AmnioMTM) is a membrane material derived from placental tissues and rich in growth factors that have been reported to have potential in healing bone. This study hypothesized that demineralized bone matrix (DBM) supplemented with AmnioMTM would accelerate healing and bone formation as compared with DBM alone in a critical size (10 mm) rat calvarial bone defect model. Five DBM grafts and 5 DBM supplemented with AmnioMTM grafts were implanted in a 10-mm critical sized defect in 10 rats (1 implant per rat). After 4 weeks, animals were euthanized and defects evaluated by microCT and histology. There were no statistical differences in microCT data for mineral density, percent bone fill, or bone surface to volume ratios between groups, though the bone surface to volume ratio for the amnio-supplemented group suggested increased osteoid activity as compared with the DBM alone group. Histological data also indicated active osteoid activity and induced bone formation in the center of defects implanted with AmnioMTM supplemented graft as compared with DBM graft alone suggesting some potential osteoinductive potential. However, there was no significant difference at the mean percent of newly mineralized bone in the DBM group defect as compared with the AmnioMTM supplemented graft material. These data suggest that while bone formation was not increased at this early time point, the increased osteoid activity and the induction of new bone in the middle of the defect by the AmnioMTM indicates that further study is needed to assess its potential benefit to bone healing and regeneration.

Hydroxyapatite scaffold pore architecture effects in large bone defects in vivo.

To examine the effect of scaffold pore size on bone regeneration within hydroxyapatite scaffolds in large segmental defects, this study evaluated two porous interconnected architectures having similar porosity and strut thickness but different pore sizes. Using a 10 mm segmental rabbit radius defect model, a bilayer scaffold architecture mimicking the cortical-cancellous organization of bone (pore size 200 µm outer layer, 450 µm inner layer) was compared to a purely trabecular-like architecture (pore size 340 µm) and an untreated defect. Bone regeneration was measured using micro-computed tomography and histology after four and eight weeks of in vivo implantation, and the mechanical strength of the defect site after eight weeks' implantation was assessed using flexural testing. Although both bilayer and trabecular architectures promoted bone growth, the trabecular scaffolds were observed to have more uniform new bone distribution within the scaffold interior at four weeks and greater bone regeneration overall after eight weeks' implantation (149 ± 9 mm³ compared to 121 ± 8 mm³ in the bilayer and 66 ± 14 mm³ in the defect). Additionally, the trabecular scaffolds were observed to exhibit significantly greater flexural strength (124% increase) and toughness (388% increase) when compared to the empty defects after eight weeks' implantation. It was concluded from this study that a larger uniform pore size led to greater functional bone regeneration over a longer implantation period for large segmental defects.

Influence of substrate curvature on osteoblast orientation and extracellular matrix deposition.

BACKGROUND: The effects of microchannel diameter in hydroxyapatite (HAp) substrates on osteoblast behavior were investigated in this study. Microchannels of 100, 250 and 500 µm diameter were created on hydroxyapatite disks. The changes in osteoblast precursor growth, differentiation, extra cellular matrix (ECM) secretion and cell attachment/orientation were investigated as a function of microchannel diameter. RESULTS: Curvature did not impact cellular differentiation, however organized cellular orientation was achieved within the 100 and 250 µm microchannels (mc) after 6 days compared to the 12 days it took for the 500mc group, while the flat substrate remained disorganized. Moreover, the 100, 250 and 500mc groups expressed a specific shift in orientation of 17.45°, 9.05°, and 22.86° respectively in 24 days. The secreted/mineralized ECM showed the 100 and 250mc groups to have higher modulus (E) and hardness (h) (E = 42.6GPa; h = 1.6GPa) than human bone (E = 13.4-25.7GPa; h = 0.47-0.74GPa), which was significantly greater than the 500mc and control groups (p < 0.05). It was determined that substrate curvature affects the cell orientation, the time required for initial response, and the shift in orientation with time. CONCLUSIONS: These findings demonstrate the ability of osteoblasts to organize and mineralize differentially in microchannels similar to those found in the osteons of compact bone. These investigations could lead to the development of osteon-like scaffolds to support the regeneration of organized bone.

Development of composite scaffolds for load-bearing segmental bone defects.

The need for a suitable tissue-engineered scaffold that can be used to heal load-bearing segmental bone defects (SBDs) is both immediate and increasing. During the past 30 years, various ceramic and polymer scaffolds have been investigated for this application. More recently, while composite scaffolds built using a combination of ceramics and polymeric materials are being investigated in a greater number, very few products have progressed from laboratory benchtop studies to preclinical testing in animals. This review is based on an exhaustive literature search of various composite scaffolds designed to serve as bone regenerative therapies. We analyzed the benefits and drawbacks of different composite scaffold manufacturing techniques, the properties of commonly used ceramics and polymers, and the properties of currently investigated synthetic composite grafts. To follow, a comprehensive review of in vivo models used to test composite scaffolds in SBDs is detailed to serve as a guide to design appropriate translational studies and to identify the challenges that need to be overcome in scaffold design for successful translation. This includes selecting the animal type, determining the anatomical location within the animals, choosing the correct study duration, and finally, an overview of scaffold performance assessment.

Guided bone regeneration in long-bone defects with a structural hydroxyapatite graft and collagen membrane.

There are few synthetic graft alternatives to treat large long-bone defects resulting from trauma or disease that do not incorporate osteogenic or osteoinductive factors. The aim of this study was to test the additional benefit of including a permeable collagen membrane guide in conjunction with a preformed porous hydroxyapatite bone graft to serve as an improved osteoconductive scaffold for bone regeneration. A 10-mm-segmental long-bone defect model in the rabbit radius was used. The hydroxyapatite scaffolds alone or with a collagen wrap were compared as experimental treatment groups to an empty untreated defect as a negative control or a defect filled with autologous bone grafts as a positive control. All groups were evaluated after 4 and 8 weeks of in vivo implantation using microcomputed tomography, mechanical testing in flexure, and histomorphometry. It was observed that the use of the wrap resulted in an increased bone volume regenerated when compared to the scaffold-only group (59% greater at 4 weeks and 27% greater after 8 weeks). Additionally, the increase in density of the regenerated bone from 4 to 8 weeks in the wrap group was threefold than that in the scaffold group. The use of the collagen wrap showed significant benefits of increased interfacial bone in-growth (149% greater) and periosteal remodeling (49%) after 4 weeks compared to the scaffold-alone with the two groups being comparable after 8 weeks, by when the collagen membrane showed close-to-complete resorption. While the autograft and wrap groups showed significantly greater flexural strength than the defect group after 8 weeks, the scaffold-alone group was not significantly different from the other three groups. It is most likely that the wrap shows improvement of function by acting like a scaffold for periosteal callus ossification, maintaining the local bone-healing environment while reducing fibrous infiltration (15% less than scaffold only at 4 weeks). This study indicates that the use of a collagen membrane with a hydroxyapatite structural graft provides benefits for bone tissue regeneration in terms of early interfacial integration.

BMP-2 tethered hydroxyapatite for bone tissue regeneration: coating chemistry and osteoblast attachment.

The goal of this study was to determine the effectiveness of using polyethyleneimine (PEI) and a polyethylene glycol (PEG) tether to bind human recombinant bone morphogenetic protein-2 (rhBMP-2) to hydroxyapatite (HAp) to enhance rhBMP-2 loading, alter its release properties, and enhance cellular interaction with the material. By using a branched PEI that was derived to express free thiols, rhBMP-2 was coated onto dense HAp surfaces at ~43 ng/cm(2). Using this novel attachment methodology, it was observed that the PEI-SH coating did not change the morphology of the HAp surfaces and that the amount of rhBMP-2 loaded was comparable to a direct adsorption method. In addition, it was also observed that the PEI and PEG tether significantly retained the rhBMP-2 to the HAp surface, inhibiting the burst release effect. Using human fetal osteoblast cells, the PEI- and PEG-tethered BMP-2 was also observed to increase cellular attachment by 10-fold when compared with uncoated HAp and adsorbed rhBMP-2. It was concluded from this study that PEI and PEG tether significantly reduce the initial burst release effect of rhBMP-2. It was also concluded that the rhBMP-2 conjugation to PEI and PEG tether promoted an increase in cellular attachment to the HAp surface.

Evaluation of BMP-2 tethered polyelectrolyte coatings on hydroxyapatite scaffolds in vivo.

The goal of this in vivo study was to evaluate the osteoinductive and angio-inductive properties of a porous hydroxyapatite (HAp) scaffold with immobilized recombinant bone morphogenetic protein-2 (rhBMP-2) on the surface. It was hypothesized in this study that the use of a rhBMP-2 incorporated polyelectrolyte coating on the HAp scaffold would allow for controlled exposure of rhBMP-2 into the tissue and would provide a sound platform for tissue growth. The scaffolds were characterized for porosity and interconnectivity using pycnometry, scanning electron microscopy and micro-ct. These scaffolds were then divided into the following four groups: (a) HAp scaffold (n-HAp group), (b) rhBMP-2 physically adsorbed on HAp scaffold (HAp-BMP-2 Group), (c) polyelectrolyte coating on HAp scaffold without rhBMP-2 (HAp-PEI Scaffold Group), and (d) polyelectrolyte coating tethered with rhBMP-2 on HAp scaffold (HAp-PEI-BMP-2 Scaffold Group). Using 18 skeletally matured New Zealand white rabbits, these scaffolds were evaluated in a nonload bearing femoral condyle plug model. The negative controls for this study have defects that were left untreated and the positive controls have defects that were filled with autologous bone graft harvested from epsilateral iliac crest. Bone induction, vessel growth, and scaffold-bone contact were analyzed after 8-week implantation using micro-CT and histomorphometry. It was concluded from this study that the use of scaffold with an attached rhBMP-2 increased the vascularization around the implant when compared with the uncoated n-HAp scaffold, a necessary step of bone regeneration. The open-pore HAp scaffold was also concluded to provide a platform for tissue growth, drug loading, and tissue interaction.

Bilayer hydroxyapatite scaffolds for maxillofacial bone tissue engineering.

PURPOSE: The frequency of alveolar ridge resorption and crestal bone loss emphasizes the clinical need for bone graft substitutes to improve local bone quality prior to dental implant placement. Microcomputed tomography has been extensively employed to estimate bone quality more objectively (ie, quantitatively) by relating it to architectural parameters. In the present study, the mechanical properties of open cellular fully interconnected bilayer hydroxyapatite scaffolds, which mimicked the cortical shell/trabecular core architecture of human bone, were investigated for suitability as bone graft substitutes for maxillofacial reconstruction. MATERIALS AND METHODS: Hydroxyapatite scaffolds with different architectures were fabricated using polymeric template pore sizes of 450 or 340 µm for the inner trabecular cores and 200 or 250 µm for the outer cortical shells in three different core-to-shell volume ratios. The architectural and mechanical properties and fluid permeability of the scaffolds were compared to reported values for maxillofacial bone. RESULTS: Whereas the elastic moduli of the scaffolds were comparable, their compressive strength was observed to be in the lower range of human mandibular trabecular bone. The microcomputed tomography architectural indices for the scaffolds were comparable to those of human trabecular bone at different locations in the human body, including the maxilla and mandible. Scaffold compressive strength, elastic modulus, and fluid conductance were 0.3 to 2.3 MPa, 40.9 to 668.1 MPa, and 8.8 to 49.9 x 10-10 m3s-1Pa-1, respectively. CONCLUSION: Open-pore bilayer scaffolds can be fabricated to exhibit sufficient mechanical integrity for maxillofacial bone graft applications to match specific bone site architecture while providing sufficient permeability to sustain bone regeneration.

Antibacterial effect and cytotoxicity of Ag-doped functionally graded hydroxyapatite coatings.

Functionally graded hydroxyapatite coatings (FGHA) doped with 1, 3, and 6.5 wt % silver (Ag) have been deposited on Titanium using ion-beam-assisted deposition. Scanning transmission electron microscopy on coating cross sections confirmed the presence of FGHA coating with mostly amorphous layers at the top and mostly crystalline layers toward the coating interface as well as the existence of 10-50 nm Ag particles distributed throughout the thickness of the coatings. Calcium release in phosphate buffered saline solution showed a high release rate of Ca at the beginning of the test, and a gradual decrease in release rate thereafter to a minimum level until day 7. Similarly, the release rate of Ag in ultra pure water was initially high in the first 4 h and then gradually decreased over a 7 days period. Antibacterial tests have shown a reduction in the viability of S. aureus in Ag-doped coatings particularly in samples with higher Ag concentrations of 3 and 6.5 wt %. Cytotoxicity tests using an osteoblast cell line, on the other hand, have demonstrated that the samples with 6.5 wt % Ag have a negative effect on osteoblast cell response, proliferation, and apoptosis as well as a negative effect on protein and osteocalcin production. It is notable that the samples with 3 wt % Ag or less presented minimal cytotoxicity compared with control surfaces. Considering both the antibacterial and cytotoxicity effects, it is suggested that the 3 wt % of Ag in FGHA coatings can be favorable.

Hydroxyapatite/polylactide biphasic combination scaffold loaded with dexamethasone for bone regeneration.

This study presents a novel design of a ceramic/polymer biphasic combination scaffold that mimics natural bone structures and is used as a bone graft substitute. To mimic the natural bone structures, the outside cortical-like shells were composed of porous hydroxyapatite (HA) with a hollow interior using a polymeric template-coating technique; the inner trabecular-like core consisted of porous poly(D,L-lactic acid) (PLA) that was loaded with dexamethasone (DEX) and was directly produced using a particle leaching/gas forming technique to create the inner diameter of the HA scaffold. It was observed that the HA and PLA parts of the fabricated HA/PLA biphasic scaffold contained open and interconnected pore structures, and the boundary between both parts was tightly connected without any gaps. It was found that the structure of the combination scaffold was analogous to that of natural bone based on micro-computed tomography analysis. Additionally, the dense, uniform apatite layer was formed on the surface of the HA/PLA biphasic scaffold through a biomimetic process, and DEX was successfully released from the PLA of the biphasic scaffold over a 1-month period. This release caused human embryonic palatal mesenchyme cells to proliferate, differentiate, produce ECM, and form tissue in vitro. Therefore, it was concluded that this functionally graded scaffold is similar to natural bone and represents a potential bone-substitute material.

Porous hydroxyapatite scaffold with three-dimensional localized drug delivery system using biodegradable microspheres.

In this study, ionic immobilization of dexamethasone (DEX)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres was performed on the hydroxyapatite (HAp) scaffold surfaces. It was hypothesized that in vivo bone regeneration could be enhanced with HAp scaffolds containing DEX-loaded PLGA microspheres compared to the use of HAp scaffolds alone. In vitro drug release from the encapsulated microspheres was measured prior to the implantation in the femur defects of beagle dogs. It was observed that porous, interconnected HAp scaffolds as well as DEX-loaded PLGA microspheres were successfully fabricated in this study. Additionally, PEI was successfully coated on PLGA microsphere surfaces, resulting in a net positive-charged surface. With such modification of the PLGA microsphere surfaces, DEX-loaded PLGA microspheres were immobilized on the negatively charged HAp scaffold surfaces. Release profile of DEX over a 4week immersion study indicated an initial burst release followed by a sustained release. In vivo evaluation of the defects filled with DEX-loaded HAp scaffolds indicated enhanced volume and quality of new bone formation when compared to defects that were either unfilled or filled with HAp scaffolds alone. This innovative platform for bioactive molecule delivery more potently induced osteogenesis in vivo, which may be exploited in implantable bone graft substitutes for stem cell therapy or improved in vivo performance. It was thus concluded that various bioactive molecules for bone regeneration might be efficiently incorporated with calcium phosphate-based bioceramics using biodegradable polymeric microspheres.

In vivo performance of bilayer hydroxyapatite scaffolds for bone tissue regeneration in the rabbit radius.

The objective of this study was to investigate the in vivo biomechanical performance of bone defects implanted with novel bilayer hydroxyapatite (HAp) scaffolds that mimic the cortical and cancellous organization of bone. The scaffolds maintained architectural continuity in a rabbit radius segmental defect model and were compared to an untreated defect group (negative control) and autologous bone grafts (positive control). Micro-CT evaluations indicated total bone and scaffold volume in the experimental group was significantly greater than the defect group but lesser than the autologous bone graft treatment. The flexural toughness of the scaffold and the autograft groups was significantly greater than the flexural toughness of the defect group. Interestingly, the absolute density of the bone mineral as well as calcium to phosphorus (Ca/P) ratio in that mineral for the scaffold and autograft contralateral bones was significantly higher than those for the defect contralaterals suggesting that the scaffolds contributed to calcium homeostasis. It was concluded from this study that new bone regenerated in the bilayer HAp scaffolds was comparable to the empty defects and while the HAp scaffolds provided significant increase in modulus when compared to empty defect and their flexural toughness was comparable to autografts after 8 weeks of implantation.

Stability of antibacterial self-assembled monolayers on hydroxyapatite.

Open fractures are common in battlefields, motor vehicle accidents, gunshot wounds, sports injuries, and high-energy falls. Such fractures are treated using hydroxyapatite (HA)-based bone graft substitutes. However, open fracture wounds are highly susceptible to bacterial infections. Hence, this study was focused on incorporating antibacterial properties to HA using silver (Ag) carrying self-assembled monolayers (SAMs). Also, the stability of Ag carrying SAMs on HA was investigated under sterilization and physiological conditions. Initially, the -COOH terminated phosphonic acid SAMs of two different chain lengths (11 carbon atoms - shorter chain and 16 carbon atoms - longer chain) were deposited on HA. Antibacterial SAMs (ASAMs) were prepared by chemically attaching Ag to shorter and longer chain SAMs coated HA. X-ray photoelectron spectroscopy, atomic force microscopy, and contact angle goniometry collectively confirmed the attachment of Ag onto SAMs coated HA. The bacterial adhesion study showed that the adherence of Staphylococcus aureus was significantly reduced on ASAMs coated HA when compared to control-HA. The stability studies showed that gas plasma, dry heat and autoclave degraded most of the ASAMs on HA. UV irradiation did not damage the shorter chain ASAMs as vigorously as other treatments, while it degraded the longer chain ASAMs completely. Ethylene oxide treatment did not degrade the longer chain ASAMs unlike all other treatments but it severely damaged the shorter chain ASAMs. Both shorter and longer chain ASAMs significantly desorbed from the HA surfaces under physiological conditions although longer chain ASAMs exhibited better stability than shorter chain ASAMs. This study demonstrated the potential for using ASAMs to provide antibacterial properties to HA and the need for developing techniques to improve stability of SAMs under sterilization and physiological conditions.

Deposition and investigation of functionally graded calcium phosphate coatings on titanium.

A series of calcium phosphate coatings with graded crystallinity were deposited onto heated titanium substrates using ion beam assisted deposition. The microstructure of the coating was examined using transmission electron microscopy (TEM). The coating thickness was observed to be in a range of 594-694 nm. The degree of crystallinity and microstructural grain size of the coating showed a clear decrease with increasing distance from the substrate-coating interface. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of PO(4)(3-), and X-ray photoelectron spectroscopy (XPS) analysis on the coating top surface showed that the atomic Ca/P ratio was in the range of 1.52+/-0.15 to 1.61+/-0.07. The biological response to the coatings was also evaluated using an osteoblast precursor cell culture test. More cells and a higher integrin expression of cell attachment sites were observed on the coating surface when compared to the control group (blank titanium surface). The pull-off test showed average adhesion strengths at the coating-substrate interface to be higher than 85.12+/-5.37 MPa. Nanoindentation tests indicated that the Young's moduli of all coatings are higher than 91.747+/-3.641 GPa and microhardness values are higher than 5.275+/-0.315 GPa. While the adhesion strength results helped us to identify the best setup for substrate temperature and processing parameters to begin the deposition, the culture test and XPS results helped identifying the optimum parameters for the last stage of deposition. TEM, X-ray diffraction, FTIR and nanoidentation results were used to further evaluate the quality of the coating and optimization of its processing parameters.