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.

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.

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.

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.

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.

In vivo study on hydroxyapatite scaffolds with trabecular architecture for bone repair.

The objective of this research was to investigate the bone formation and angio-conductive potential of hydroxyapatite (HA) scaffolds closely matched to trabecular bone in a canine segmental defect after 3 and 12 weeks post implantation. Histomorphometric comparisons were made between naturally forming trabecular bone (control) and defects implanted with scaffolds fabricated with micro-size (M-HA) and nano-size HA (N-HA) ceramic surfaces. Scaffold architecture was similar to trabecular bone formed in control defects at 3 weeks. No significant differences were identified between the two HA scaffolds; however, significant bone in-growth was observed by 12 weeks with 43.9 +/- 4.1% and 50.4 +/- 8.8% of the cross-sectional area filled with mineralized bone in M-HA and N-HA scaffolds, respectively. Partially organized, lamellar collagen fibrils were identified by birefringence under cross-polarized light at both 3 and 12 weeks post implantation. Substantial blood vessel infiltration was identified in the scaffolds and compared with the distribution and diameter of vessels in the surrounding cortical bone. Vessels were less numerous but significantly larger than native cortical Haversian and Volkmann canals reflecting the scaffold architecture where open spaces allowed interconnected channels of bone to form. This study demonstrated the potential of trabecular bone modeled, highly porous and interconnected, HA scaffolds for regenerative orthopedics.

Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration.

To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.

Ultrasound effect on osteoblast precursor cells in trabecular calcium phosphate scaffolds.

This study investigated the in vitro effect of low-intensity pulsed ultrasound (LIPUS) on human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an osteoblast precursor cell line, during early adhesion to calcium phosphate scaffolds. Hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) ceramic scaffolds were produced by a template coating method. Phospho-specific antibody cell-based ELISA (PACE) technique was utilized on stress activation proteins, including the extracellular signal-regulated kinase (ERK1/2), P38, c-Jun N-terminal kinase (JNK) and the anti-apoptosis mediator protein kinase B (PKB/AKT). Cell-based ELISAs were also performed on the membrane anchoring protein vinculin and alpha6beta4 integrin. LIPUS stimulated activation of PERK 1/2, PJNK, PP38 and vinculin in traditional two-dimensional (2-D) culture. Calcium release from the scaffolds was partially involved in the activation of PERK 1/2 when cell response was compared between culture on 2-D surfaces and three-dimensional (3-D) HA and TCP scaffolds. Effects of calcium extracted media from scaffolds alone could not account for the full activation of PJNK, PP38, PAKT, vinculin and alpha6beta4 integrin. LIPUS stimulation further increased PERK activity on TCP scaffolds corresponding with an increase in both vinculin and alpha6beta4 integrin levels. It was concluded from this study that LIPUS treatment can significantly affect stress signaling mediators and adhesion proteins in osteoblast precursor cells during the early cell-attachment phase to trabecular patterned scaffolds.

Effects of trabecular calcium phosphate scaffolds on stress signaling in osteoblast precursor cells.

The objective of this research was to investigate stress-signaling patterns in response to two-dimensional (2-D) and three-dimensional (3-D) calcium phosphate (CP) materials using human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an osteoblast precursor cell line. Control discs and scaffolds were fabricated from hydroxyapatite and beta tri-CP ceramics. Phospho-specific antibody cell-based ELISA technique was utilized on members of the mitogen-activated protein kinase cascade including; the extracellular signal-regulated kinases (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and the anti-apoptosis mediator protein kinase B (AKT). Quantification of these signals was evaluated during the early attachment phase of osteoblast precursor cells. In this study, it was observed that 3-D CP scaffolds significantly activated the stress mediators p38 and JNK but not ERK1/2. This signal trend was matched with an up-regulation in AKT, suggesting the ability of cells to manage high stress signals in response to 3-D CP architecture and that 3-D CP scaffolds are necessary for studies simulating a natural trabecular bone organization. The absence of these signals in 2-D CP surfaces indicated the importance of local architecture conditions on cell stress response. It was concluded from this study that osteoblast precursor cells cultured in 3-D CP scaffolds experience greater stress-signaling patterns when compared to 2-D CP surfaces.