Advances in Controlled Drug Delivery for Treatment of Osteoporosis.

Osteoporosis, which is characterized by resorption of bone exceeding formation, remains a significant human health concern, and the impact of this condition will only increase with the "graying" of the worldwide population. This review focuses on current and emerging approaches for delivering therapeutic agents to restore bone remodeling homeostasis. Well-known antiresorptive and anabolic agents, such as estrogen, estrogen analogs, bisphosphonates, calcitonin, and parathyroid hormone, along with newer modulators and antibodies, are primarily administered orally, intravenously, or subcutaneously. Although these treatments can be effective, continuing problems include patient noncompliance and adverse systemic or remote-site effects. Controlled drug delivery via polymeric, targeted, and active release systems extends drug half-life by shielding against premature degradation and improves bioavailability while also providing prolonged, sustained, or intermittent release at therapeutic doses to more effectively treat osteoporosis and associated fracture risk.

In vitro evaluation of osteoblast responses to carbon nanotube-coated titanium surfaces.

BACKGROUND: The effects of surface roughness and carboxyl functionalization of multi-walled carbon nanotubes (MWCNTs) mixed with collagen coated onto titanium (Ti) substrates on MC3T3-E1 osteoblasts were evaluated. METHODS: The proliferation, differentiation, and matrix mineralization were investigated using (1) smooth-surfaced Ti discs, (2) Ti discs coated with collagen and MWCNT (Ti-MWCNT), and (3) Ti discs coated with collagen and MWCNT-COOH (Ti-MWCNT-COOH) for applications in orthodontic mini screw implants (MSIs). The coatings were uniform when analyzed using scanning electron microscopy (SEM), and surface roughness was evaluated by surface profilometry that demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 µm) and MWCNT-COOH (0.84 ± 0.01 µm) groups. MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assay was performed after days 1, 3, and 7 to assess proliferation. Alkaline phosphatase (ALP)-specific activity was assessed after day 7 to quantify differentiation. Alizarin red staining was measured after day 28 to quantify matrix mineralization. All data were analyzed with JMP Pro11 software (SAS, USA) with a statistical significance of p < 0.05. RESULTS: Surface profilometry demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 µm) and MWCNT-COOH (0.84 ± 0.01 µm) groups. On day 7, ALP assay showed that MWCNT-COOH (mean ± SD 0.98 ± 0.26 U/µg of protein) enhanced cell differentiation when compared to the uncoated group (p = 0.05). Alizarin red staining after 28 days of cell culture revealed that MWCNT-COOH (mean ± SD 1.5 ± 0.2 OD405) increased (p = 0.03) matrix mineralization when compared to the uncoated group (0.9 ± 0.09 OD405). CONCLUSIONS: This study showed that coatings containing MWCNT-COOH (increased hydrophilic surface chemistry) influence osteoblast proliferation, differentiation, and matrix mineralization and should be further studied for applications in orthodontic MSIs.

Porous PLGA scaffolds for controlled release of naked and polyethyleneimine-complexed DNA.

The ability to precisely control delivery of single or multiple bioactive molecules is critical in tissue engineering, and controlled release of plasmid coding for growth factors and their regulators can give cell-regulated, short-term expression of these therapeutic biomolecules. In this work, porous poly(lactic-co-glycolic acid) (PLGA) scaffolds comprising acid-terminated chains of either low (LMW; 10 kDa) or high molecular weight (HMW; 30 kDa) were developed for controlled release of naked or polyethyleneimine (PEI)-complexed DNA. The compressive strength of blank HMW and LMW scaffolds was 6 and 2 MPa, respectively, while the strength of PEI:DNA-containing HMW and LMW scaffolds was 7 and 1 MPa, respectively. LMW scaffolds degraded more quickly than HMW scaffolds, with 80-100% and 15-30% mass loss at 30 days, respectively. Encapsulation of plasmid, particularly PEI-complexed DNA, only modestly affected degradation. Release profiles showed bi- or triphasic patterns, with early burst release of surface-associated DNA, slower diffusion-mediated release, and degradation-related release at later time points. Complexation with PEI tended to a slow release of plasmids, likely because of interaction with the carboxyl groups of PLGA. Culturing rat bone marrow cells on blank PLGA scaffolds in the presence of IGF-I resulted in growth and chondrogenic differentiation of these cells. Porous scaffolds made of PLGA with the appropriate selection of hydrophobicity and molecular weight will allow controlled delivery of naked and condensed plasmid DNA for different tissue engineering applications.

Development of an injectable two-phase drug delivery system for sequential release of antiresorptive and osteogenic drugs.

Unlike controlled release systems that deliver a single drug, dual or multidrug delivery systems with distinct release profiles are more likely to promote timely and effective tissue regeneration as they provide both temporally and concentration-dependent release of different molecules to mimic natural biological events. In this study, an injectable and biodegradable delivery system was developed to sequentially release an antiresorptive drug (clodronate) followed by an osteogenic agent (simvastatin) to treat bone disease. The injectable delivery system comprised simvastatin-loaded gelatin microspheres suspended in a viscous solution of carboxymethylcellulose (CMC) containing clodronate. Several factors (CMC concentration, glutaraldehyde concentration, simvastatin loading, and gelatin microsphere processing conditions) were investigated for their effects on drug release. Clodronate release was not affected by CMC concentration, with complete delivery within 12 hr, and simvastatin release could be modulated by cross-linking of the gelatin microspheres, loading, and washing conditions. Burst release of simvastatin was reduced from 70% to 6% in conjunction with sustained release for up to 3 weeks. The combined system showed early release of the antiresorptive clodronate sequentially followed by sustained delivery of the osteogenic simvastatin. This robust and flexible two-phase delivery system may prove useful for applications in which multiple drug delivery is desired.

Bioactivity of WLBU2 peptide antibiotic in combination with bioerodible polymer.

WLBU2 is a peptide antibiotic designed for broad antimicrobial activity, including bacteria associated with periodontal disease. Although periodontitis is associated with various systemic conditions, ranging from cardiovascular disease to preterm birth, local therapy is needed to treat the source of infection. Biodegradable polymers are often used to control locally the amount and rate of delivery of drugs. In the present study, a bioerodible association polymer comprising cellulose acetate phthalate (CAP) and Pluronic F-127 (PF-127) was explored for its interaction with WLBU2. The intrinsic antimicrobial activity of CAP/PF-127 and the combined effects of the polymer and WLBU2 were examined using Streptococcus gordonii, a species involved in early colonisation of tooth surfaces. The polymer blend alone had dose-dependent bacteriostatic properties, resulting in a ≥ 2 log decrease in colonies at the highest concentrations tested, possibly due to the hydrophobicity of CAP disrupting the surface of bacteria. When WLBU2 was combined with CAP/PF-127, an apparent binding of peptide to polymer significantly decreased the activity compared with free WLBU2, which functions like other cationic peptides by destabilising the bacterial membrane. Formulation with sucrose as an excipient, which reduced the interaction between WLBU2 and polymer, restored the bactericidal activity of the peptide antibiotic as reflected by a > 3 log decrease in S. gordonii. WLBU2 can be locally delivered using CAP/PF-127 as a release vehicle, with the peptide's bactericidal activity dominating the polymer's bacteriostatic effect.

Infection, inflammation, and bone regeneration: a paradoxical relationship.

Various strategies have been developed to promote bone regeneration in the craniofacial region. Most of these interventions utilize implantable materials or devices. Infections resulting from colonization of these implants may result in local tissue destruction in a manner analogous to periodontitis. This destruction is mediated via the expression of various inflammatory mediators and tissue-destructive enzymes. Given the well-documented association among microbial biofilms, inflammatory mediators, and tissue destruction, it seems reasonable to assume that inflammation may interfere with bone healing and regeneration. Paradoxically, recent evidence also suggests that the presence of certain pro-inflammatory mediators is actually required for bone healing. Bone injury (e.g., subsequent to a fracture or surgical intervention) is followed by a choreographed cascade of events, some of which are dependent upon the presence of pro-inflammatory mediators. If inflammation resolves promptly, then proper bone healing may occur. However, if inflammation persists (which might occur in the presence of an infected implant or graft material), then the continued inflammatory response may result in suboptimal bone formation. Thus, the effect of a given mediator is dependent upon the temporal context in which it is expressed. Better understanding of this temporal sequence may be used to optimize regenerative outcomes.

Formulations for intermittent release of parathyroid hormone (1-34) and local enhancement of osteoblast activities.

The objective of these studies was to develop simple, implantable devices that intermittently release PTH(1-34) and thus could be used for locally stimulating bone formation. The formulations were based on the association polymer system of cellulose acetate phthalate and Pluronic F-127. Release profiles for intermittent devices showed five discrete peaks, whereas sustained devices exhibited zero-order kinetics. Osteoblastic activity was greater for cells intermittently treated with PTH(1-34) compared to sustained exposure. These controlled release devices delivering PTH(1-34) in an intermittent manner may be useful for affecting osteoblast activities in a localized area.

Immobilization of glycoproteins, such as VEGF, on biodegradable substrates.

Attachment of growth factors to biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA), may enhance and/or accelerate integration of tissue engineering scaffolds. Although proteins are commonly bound via abundant amino groups, a more selective approach may increase bioactivity of immobilized molecules. In this research, exposed carboxyl groups on acid-terminated PLGA were modified with dihydrazide spacer molecules. The number of hydrazide groups available for subsequent attachment of protein was dependent on dihydrazide length, with shorter molecules present at significantly greater surface densities. The potent angiogenic glycoprotein vascular endothelial growth factor (VEGF) was oxidized with periodate and the aldehyde moieties allowed to react with the hydrazide-derivatized PLGA. Derivatization initially affected the amount of protein bound to the surfaces, but differences were substantially reduced following overnight incubation in saline. More importantly, use of shorter dihydrazide spacers significantly enhanced accessibility of immobilized VEGF for binding neutralizing antibody and soluble VEGF receptor. Furthermore, immobilized growth factor enhanced endothelial cell proliferation, with surfaces having the shortest and longest spacers stimulating greater effects. The present work has not only demonstrated an alternative approach to immobilizing growth factors on biodegradable materials, but the scheme can be used to alter the amount of protein bound as well as its availability for subsequent biointeractions.

Protein Binding to Peptide-Imprinted Porous Silica Scaffolds.

Of the many types of biomolecules used for molecular imprinting applications, proteins are some of the most useful, yet challenging, templates to work with. One method, termed the 'epitope approach', involves imprinting a short peptide fragment of the protein into the polymer to promote specific adsorption of the entire protein, similar to the way an antigen binds to an antibody via the epitope. Whole lysozyme or the 16 residue lysozyme C peptide was imprinted into porous silica scaffolds using sol-gel processing. After removing template, scaffolds were exposed to lysozyme and/or RNase A, which was used as a competitor molecule of comparable size. When comparing protein- to peptide-imprinted scaffolds, similar amounts of lysozyme and RNase were bound from single protein solutions. However, while whole lysozyme-imprinted scaffolds showed about 4:1 preferential binding of lysozyme to RNase, peptide-imprinted scaffolds failed to show statistical significance, even though a slight preferential binding trend was present. These initial studies suggest there is potential for using peptide-imprinting to create specific protein-binding sites on porous inorganic surfaces, although further development of the materials is needed.

Protein-imprinted polysiloxane scaffolds.

Molecular imprinting is a technique used to create specific recognition sites on the surface of materials. Although widely developed for chromatographic separation of small molecules, this approach has not been adequately investigated for biomaterial applications. Thus, the objective of these experiments was to explore the potential of molecular imprinting for creating biomaterials that preferentially bind specific proteins. Macroporous polysiloxane (silica) scaffolds were imprinted with either lysozyme or RNase A using sol-gel processing. The quantity of surface-accessible protein, which was related to the number of potential binding sites, was varied by changing the amount of protein loaded into the sol. Up to 62% of loaded protein was accessible. The amount of protein per unit surface area ranged from 0.3microgm(-2) for low loading of RNase to 152microgm(-2) for high loading of lysozyme. Protein-imprinted scaffolds were then evaluated for their ability to preferentially recognize the template biomolecule when incubated in mixtures containing both the imprinted protein and a competitor protein of comparable size (approximately 14kD). In solutions containing a single protein, up to 3.6 times more template bound compared with the competitor. Furthermore, in solutions containing equal amounts of both molecules, the porous scaffolds bound up to three times more template than the competitor protein, which is a level of preferential binding similar to values reported in the molecular imprinting literature for both organic and inorganic materials.

Mechanical and degradation behavior of polymer-calcium sulfate composites.

Calcium sulfate (CS) is one of the oldest bone graft materials still in use. Its main limitations are poor handling characteristics, poor mechanical properties, and a resorption rate that is too fast for some applications. The present study investigated the effect of viscous polymers, such as carboxymethylcellulose (CMC) and hyaluronan (HY), on the handling characteristics, mechanical properties, and degradation behavior of CS. CMC and HY were added to CS at concentrations from 1-10 wt%. Addition of CMC to CS at more than 4 wt% produced a putty-like material and decreased the density of the composite, while also increasing flexural and compressive strength at higher loadings. Incorporation of CMC produced a concentration-dependent increase in water absorption and degradation rate. At an equivalent loading, HY-containing CS composites showed better compressive strength than CS with CMC. Overall, addition of CMC or HY to CS resulted in composite materials with better handling characteristics and improved mechanical properties after set, however the degradation rate of the augmented materials was increased. These properties suggest that the enhanced CS materials may be useful in certain clinical situations, such as filling non-uniform bone defects and situations that require mechanical integrity of the bone graft substitute during implantation.

Modulated release of bioactive protein from multilayered blended PLGA coatings.

The objective of this study was to develop a poly(D,L-lactic-co-glycolic acid) (PLGA)-based coating system for producing biologically-inspired delivery profiles. Protein-loaded microspheres were made from PLGA (50:50) terminated with carboxylic acid groups (PLGA-2A) blended either with more hydrophobic PLGA (50:50) having lauryl ester endcaps (PLGA-LE) or with the more hydrophilic Pluronic F-127 (PF-127). Dense coatings were formed by pressure-sintering the microspheres. Altering hydrophobicity changed the water concentration within coatings, and consequently the time to onset of polymer degradation and protein release was modulated. After blending up to 8% Pluronic, degradation by-products began accumulating immediately upon incubation in saline, whereas, degradation was delayed for up to 14 days with blending of up to 30% PLGA-LE. Primary protein release peaks from one-layer coatings could be created from 7 to 20 days using 8% PF-127 or 30% PLGA-LE blends, respectively. Multilayered coatings of different blends generated several release peaks, with their temporal occurrence remaining approximately the same when layers of other hydrophobicity were added above or below. To allow design of coatings for future use, results were used to construct a model based on Fourier analysis. This polymer blend system and model can be used to mimic temporally varying profiles of protein expression.

Cell responses to BMP-2 and IGF-I released with different time-dependent profiles.

During wound healing, growth factors are expressed in time-dependent amounts. Constant delivery of biomolecules, however, is often used to influence cell and tissue behavior. In the present studies, a crosslinked gelatin-coating system was used to deliver bone morphogenetic protein 2 (BMP-2) or insulin-like growth factor (IGF-I) to three types of mesenchymal cells with three temporally varying release profiles. The "early" delivery profile released most of the growth factor within the first 2 days. The "pseudo-zero-order" profile approximated constant rate of delivery for about 5 days. The "late" delivery profile released most of the growth factor after about 5 days. Early delivery of IGF-I had the greatest effect on mitogenesis of SaOS-2 human osteosarcoma cells with a secondary effect noted nearly 5 days after delivery was completed. Late delivery of BMP-2 resulted in greatest alkaline phosphatase (AP) activity in mouse pluripotent C3H10T1/2 cells. Rat bone marrow stromal cells (BMCs) responded to all delivery profiles of BMP-2, with the duration of elevated AP activity increasing as the amount of BMP-2 delivered increased. In addition to an early increase in AP activity, late release also stimulated BMCs over a longer portion of the culture period. BMCs responded similarly to SaOS-2 cells when seeded on early IGF-I delivery coatings, increasing AP activity after delivery had ended. Overall, these studies further show the importance of delivery profile, specifically the characteristics of time and concentration, on cell and tissue responses.

In vitro effects of combined and sequential delivery of two bone growth factors.

Bone formation and repair occur by a complex cascade involving numerous growth factors and cytokines. In this study, two-layered heterogeneously loaded and crosslinked gelatin coatings were used to obtain combined and sequential delivery of two bone growth factors, BMP-2 and IGF-I, in cell cultures. Peak release from the top and bottom layers was localized around 1 and 6 days, respectively. For comparison, cells were also treated with soluble growth factors directly added to the culture medium. Pluripotent C3H10T1/2 (C3H) cells responded to soluble growth factor treatments with the greatest specific alkaline phosphatase (AP) activity resulting from addition of BMP-2 followed by IGF-I or by BMP-2+IGF-I. Altered loading and subsequent release of BMP-2 and IGF-I from gelatin coatings also affected AP activity in C3H cultures, and the coatings influenced AP activity and incorporation of calcium in the extracellular matrix of bone marrow stromal cell cultures. Early delivery of BMP-2 followed by increased release of BMP-2 and IGF-I after 5 days resulted in the largest, as well as earliest, elevation of AP activity and mineralized matrix formation compared to controls and other treatments. Simultaneous release of both growth factors from both layers did not significantly change AP activity or matrix calcium content compared to control coatings. These results demonstrate that temporally varying delivery of multiple growth factors can significantly affect cell behavior.

A technique to immobilize bioactive proteins, including bone morphogenetic protein-4 (BMP-4), on titanium alloy.

Immobilization of biomolecules on surfaces enables both localization and retention of molecules at the cell-biomaterial interface. Since metallic biomaterials used for orthopedic and dental implants possess a paucity of reactive functional groups, biomolecular modification of these materials is challenging. In the present work, we investigated the use of a plasma surface modification strategy to enable immobilization of bioactive molecules on a "bioinert" metal. Conditions during plasma polymerization of allyl amine on Ti-6Al-4V were varied to yield 5 ("low")- and 12 ("high")-NH2/nm2. One- and two-step carbodiimide schemes were used to immobilize lysozyme, a model biomolecule, and bone morphogenetic protein-4 (BMP-4) on the aminated surfaces. Both schemes could be varied to control the amount of protein bound, but the one-step method destroyed the activity of immobilized lysozyme because of crosslinking. BMP-4 was then immobilized using the two-step scheme. Although BMP bound to both low- and high-NH2 surfaces was initially able to induce alkaline phosphatase activity in pluripotent C3H10T1/2 cells, only high amino group surfaces were effective following removal of weakly bound protein by incubation in cell culture medium.

Triphasic release model for multilayered gelatin coatings that can recreate growth factor profiles during wound healing.

Multilayered gelatin coatings were created to mimic growth factor profiles that normally occur during fracture healing. A model was developed to relate crosslinking and loading of individual layers to protein release. Modeling was simplified by dividing release profiles into three phases. The diffusion-controlled phase was determined by calculating periods of constant diffusivity for each homogeneous layer within devices. Diffusivity was a power law function of crosslinking. Fick's second law of diffusion was then used to determine release during the diffusion-controlled phase. Secondary diffusivity was determined by summing resistances of each successive homogeneous layer. The initial burst phase was defined as events proceeding the diffusion-controlled phase. Percentage of drug burst was a linear function of crosslinking. Release during the degradation-controlled phase, events following diffusion-controlled phase, was estimated based on first order hydrolysis of crosslinks. The model predicted time-variant release of differently labeled protein measured experimentally, and it can be used to design coatings to recreate the cascade of biomolecules that determine natural bone repair.

Biomaterials and biomechanics of oral and maxillofacial implants: current status and future developments.

Research in biomaterials and biomechanics has fueled a large part of the significant revolution associated with osseointegrated implants. Additional key areas that may become even more important--such as guided tissue regeneration, growth factors, and tissue engineering--could not be included in this review because of space limitations. All of this work will no doubt continue unabated; indeed, it is probably even accelerating as more clinical applications are found for implant technology and related therapies. An excellent overall summary of oral biology and dental implants recently appeared in a dedicated issue of Advances in Dental Research. Many advances have been made in the understanding of events at the interface between bone and implants and in developing methods for controlling these events. However, several important questions still remain. What is the relationship between tissue structure, matrix composition, and biomechanical properties of the interface? Do surface modifications alter the interfacial tissue structure and composition and the rate at which it forms? If surface modifications change the initial interface structure and composition, are these changes retained? Do surface modifications enhance biomechanical properties of the interface? As current understanding of the bone-implant interface progresses, so will development of proactive implants that can help promote desired outcomes. However, in the midst of the excitement born out of this activity, it is necessary to remember that the needs of the patient must remain paramount. It is also worth noting another as-yet unsatisfied need. With all of the new developments, continuing education of clinicians in the expert use of all of these research advances is needed. For example, in the area of biomechanical treatment planning, there are still no well-accepted biomaterials/biomechanics "building codes" that can be passed on to clinicians. Also, there are no readily available treatment-planning tools that clinicians can use to explore "what-if" scenarios and other design calculations of the sort done in modern engineering. No doubt such approaches could be developed based on materials already in the literature, but unfortunately much of what is done now by clinicians remains empirical. A worthwhile task for the future is to find ways to more effectively deliver products of research into the hands of clinicians.

Understanding and controlling the bone-implant interface.

A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, endosseous implants should result in formation of a characteristic interfacial layer and bone matrix with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the interface and of the effects biomaterials have on bone and bone cells is needed. Such knowledge is essential for developing strategies to optimally control osseointegration. This paper reviews current knowledge of the bone-biomaterial interface and methods being investigated for controlling it. Morphological studies have revealed the heterogeneity of the bone-implant interface. One feature often reported, regardless of implant material, is an afibrillar interfacial zone, comparable to cement lines and laminae limitantes at natural bone interfaces. These electron-dense interfacial layers are rich in noncollagenous proteins, such as osteopontin and bone sialoprotein. Several approaches, involving alteration of surface physicochemical, morphological, and/or biochemical properties, are being investigated in an effort to obtain a desirable bone-implant interface. Of particular interest are biochemical methods of surface modification, which immobilize molecules on biomaterials for the purpose of inducing specific cell and tissue responses or, in other words, to control the tissue-implant interface with biomolecules delivered directly to the interface. Although still in its infancy, early studies indicate the value of this methodology for controlling cell and matrix events at the bone-implant interface.

Release and retention of biomolecules in collagen deposited on orthopedic biomaterials.

Delivery of osteotropic biomolecules directly to the bone-implant interface can alter initial interactions between tissue and biomaterial. To this end, type I collagen coatings containing a model biomolecule, lysozyme, were deposited on Co-Cr-Mo and Ti-6Al-4V. Two deposition methods were examined. In the first, lysozyme was deposited concurrently with collagen, while in the second, protein was impregnated into previously deposited collagen coatings. The amount of collagen and the amount of lysozyme loaded into collagen were varied to provide different amounts of weakly and strongly bound protein. Release and retention of lysozyme were monitored over a 7 d period of incubation in physiological saline. For both methods, larger amounts of collagen in the coatings allowed incorporation of more lysozyme. Additionally, loading collagen coatings with greater amounts of lysozyme resulted in release of more protein. During the first 24-96 h of incubation, loosely bound protein was eluted, resulting in release of 2 micrograms to 55 mg (5-75% of the amount available) of enzymatically active lysozyme. This left 25-95% of the protein bound to the collagen-coated biomaterials and, thus, available for later release during degradation of the collagen.

In vitro cellular responses to bioerodible particles loaded with recombinant human bone morphogenetic protein-2.

Porous 50:50 poly(d,l lactide-co-glycolide) microspheres containing varying amounts of "free" recombinant human bone morphogenetic protein-2 (rhBMP-2) were evaluated for their ability to induce/enhance expression of osteoblastic characteristics by pluripotent mesenchymal cells in vitro. "Free" protein (Fp) is defined as protein present on the surface and within the porous matrix of the microspheres. Four preparations of bioerodible particles (BEP) were used: blank--without rhBMP-2; low Fp--24 microg of free rhBMP-2 per g of particles; medium Fp--403 microg/g; and high Fp--884 microg/g. C3H10T1/2 cells (C3H) and bone marrow stromal cells (BMC) were cultured with 1 mg of BEP for up to 4 weeks, and cell growth and expression of osteogenic responses were determined weekly. For both cell types, control cultures (neither BEP nor rhBMP-2) and cultures with blank BEP exhibited no or minimal osteoblastic characteristics. Compared to control and blank BEP cultures, C3H cells responded to particles having medium and high amounts of free rhBMP-2 with increased cell growth and alkaline phosphatase activity, but osteocalcin secretion and mineralization were not markedly influenced. Low Fp BEP enhanced only the alkaline phosphatase activity of C3H cells. In contrast, although growth was not affected, rhBMP-2-loaded BEP increased alkaline phosphatase activity, osteocalcin secretion, and mineralization in BMC cultures in a dose-dependent manner (i.e., blank < low < medium < high Fp).