Synthesis of poly(meth)acrylates with thioether and tertiary sulfonium groups by ARGET ATRP and their use as siRNA delivery agents.

The field of RNA interference depends on the development of safe and efficient carriers for short interfering ribonucleic acid (siRNA) delivery. Conventional cationic monomers for siRNA delivery have utilized the nitrogen heteroatom to produce cationic charges. Here, we polymerized cationic sulfonium (meth)acrylate by activators regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) to form polymers with narrow molecular weight distributions for siRNA delivery. The tertiary sulfonium species was stable toward dealkylation in water but less stable in the polar aprotic solvent dimethyl sulfoxide. Block copolymers poly(ethylene oxide) with poly(meth)acrylate containing sulfonium moieties were prepared as an siRNA delivery platform. Results suggested block copolymers were biocompatible up to 50 µg/mL in vitro and formed polyplexes with siRNA. Additionally, block copolymers protected siRNAs against endonuclease digestion and facilitated knockdown of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) mRNA expression in murine calvarial preosteoblasts. The versatility, biocompatibility, and cationic nature of these tertiary sulfonium groups are expected to find widespread biological applications.

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells.

BACKGROUND: Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation. QUESTIONS/PURPOSES: We hypothesized that (1) nanogel-mediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures. METHODS: Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage™ assay). RESULTS: Gene expression assays demonstrated that nanogel-based RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p < 0.001) and 43.22% ± 18.01% (both p < 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p < 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p < 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that nanogel 1:1, 5:1, and 10:1 RNAi treatments decreased mineralization (ie, HA deposition) from cultures treated only with rhBMP-2 (p < 0.001). However, despite RNAi attack on Runx2 and Osx, HA deposition levels remained greater than non-rhBMP-2-treated cell cultures. CONCLUSIONS: Although mRNA and protein knockdown were confirmed as a result of RNAi treatments against Runx2 and Osx, complete elimination of mineralization processes was not achieved. RNAi targeting mid- and late-stage osteoblast differentiation markers such as ALP, osteocalcin, osteopontin, and bone sialoprotein) may produce the desired RNAi-nanogel nanostructured polymer HO prophylaxis. CLINICAL RELEVANCE: Successful HO prophylaxis should target and silence osteogenic markers critical for heterotopic bone formation processes. The identification of such markers, beyond RUNX2 and OSX, may enhance the effectiveness of RNAi prophylaxes for HO.

Preparation of cationic nanogels for nucleic acid delivery.

Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.

Growth factors and craniofacial surgery.

The specialty of craniofacial surgery is broad and includes trauma, aesthetics, reconstruction of congenital deformities, and regeneration of tissues. Moreover, craniofacial surgery deals with a diverse range of tissues including both "soft" and "hard" tissues. Technological advances in materials and biological sciences and improved surgical techniques have remarkably improved clinical outcomes. The quest to raise the bar for patient care continues to inspire advances for predictable biological regeneration of soft and hard tissues. As a consequence of this quest for advancement, a wide spectrum of biologicals is becoming available to surgeons. Is the use of recombinant DNA engineered biologicals daring? Sensible? Logical? Timely? Safe? It is crucial for the practicing craniofacial surgeon to take a step back periodically and carefully review the biological factors that have the potential for dramatically altering the discipline of craniofacial surgery. With this emphasis, the coauthors of this article will focus on growth factor technology underscoring bone tissue regeneration. As the 21st-century matures, recombinant human biologicals will have an overwhelming impact on the practice of craniofacial surgery.

Osteogenic differentiation of pre-osteoblasts on biomimetic tyrosine-derived polycarbonate scaffolds.

The osteogenic potential of biomimetic tyrosine-derived polycarbonate (TyrPC) scaffolds containing either an ethyl ester or a methyl ester group combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) was assessed using the preosteoblast cell line MC3T3-E1. Each composition of TyrPC was fabricated into 3D porous scaffolds with a bimodal pore distribution of micropores <20 µm and macropores between 200 and 400 µm. Scanning electron microscopy (SEM) characterization suggested MC3T3-E1 cell attachment on the TyrPC scaffold surface. Moreover, the 3D TyrPC-containing ethyl ester side chains supported osteogenic lineage progression, alkaline phosphatase (ALP), and osteocalcin (OCN) expression as well as an increase in calcium content compared with the scaffolds containing the methyl ester group. The release profiles of rhBMP-2 from the 3D TyrPC scaffolds by 15 days suggested a biphasic rhBMP-2 release. There was no significant difference in bioactivity between rhBMP-2 releasate from the scaffolds and exogenous rhBMP-2. Lastly, the TyrPC containing rhBMP-2 promoted more ALP activity and mineralization of MC3T3-E1 cells compared with TyrPC without rhBMP-2. Consequently, the data strongly suggest that TyrPC scaffolds will provide a highly useful platform for bone tissue engineering.

Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery.

Star polymers with poly(ethylene glycol) (PEG) arms and a degradable cationic core were synthesized by the atom transfer radical copolymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate macromonomer (PEGMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a disulfide dimethacrylate (cross-linker, SS) via an "arm-first" approach. The star polymers had a diameter ~15 nm and were degraded under redox conditions by glutathione treatment into individual polymeric chains due to cleavage of the disulfide cross-linker, as confirmed by dynamic light scattering. The star polymers were cultured with mouse calvarial preosteoblast-like cells, embryonic day 1, subclone 4 (MC3T3-E1.4) to determine biocompatibility. Data suggest star polymers were biocompatible, with ≥ 80% cell viability after 48 h of incubation even at high concentration (800 µg/mL). Zeta potential values varied with N/P ratio confirming complexation with siRNA. Successful cellular uptake of the star polymers in MC3T3-E1.4 cells was observed by confocal microscopy and flow cytometry after 24 h of incubation.

Photo-cross-linkable thermoresponsive star polymers designed for control of cell-surface interactions.

Star polymers with thermoresponsive arms, consisting of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate with ~4 ethylene oxide units (OEOMA300, M(n) = 300), were synthesized via atom transfer radical polymerization (ATRP). 25% of the arms contained benzophenone chain-end functionality at the star periphery. A mixture of linear poly(MEO2MA-co-OEOMA300)-Br macroinitiators without and with benzophenone end-group macroinitiators were (MI and Bzp-MI, respectively) cross-linked with ethylene glycol dimethacrylate to form star polymers. Formation of star polymers was monitored by GPC, and the presence of benzophenone functionality in the stars was confirmed by ¹H NMR. The UV-vis spectroscopy revealed that the star polymers exhibit the low critical solution temperature (LCST) at 27 °C, slightly lower than LCST of either MI or Bzp-MI. Commercially available tissue culture grade polystyrene surface was modified by depositing a thin film of functionalized stars and UV cross-linking (λ = 365 nm). The star polymers covalently attached onto surfaces allowed a control of cell shrinkage and attachment in response to temperature changes.

Rapid cellular internalization of multifunctional star polymers prepared by atom transfer radical polymerization.

Poly(ethylene glycol) (PEG) star polymers containing GRGDS (Gly-Arg-Gly-Asp-Ser) peptide sequences on the star periphery were synthesized by atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMA), GRGDS modified poly(ethylene glycol) acrylate (GRGDS-PEG-Acryl), fluorescein o-methacrylate (FMA), and ethylene glycol dimethacrylate (EGDMA) via an "arm-first" method. Star polymers were approximately 20 nm in diameter, as measured by dynamic light scattering and atomic force microscopy. Conjugation of FMA to the stars was confirmed by fluorescence microscopy, and successful attachment of GRGDS segments to the star periphery was confirmed by (1)H NMR spectroscopy. Both fluorescent PEG star polymers with and without peripheral GRGDS peptide segments were cultured with MC3T3-E1.4 cells. These star polymers were biocompatible with ≥ 90% cell viability after 24 h of incubation. Cellular uptake of PEG star polymers in MC3T3-E1.4 cells was observed by confocal microscopy. Rapid uptake of PEG star polymers with GRGDS peptides (∼ 100% of FITC-positive cells in 15 min measured by flow cytometry) was observed, suggesting enhanced delivery potential of these functional star polymers.

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.

Cell-adhesive star polymers prepared by ATRP.

This study presents the synthesis and evaluation of cell adhesive poly(ethylene oxide) (PEO) star polymers for potential biomedical applications. Star polymers with a size of approximately 20 nm and with relatively low polydispersities (M(w)/M(n) ≤ 1.6), containing GRGDS (Gly-Arg-Gly-Asp-Ser) segments, were prepared by atom transfer radical copolymerization of PEO methyl ether methacrylate macromonomer (MM), telechelic GRGDS-PEO-acrylate MM, and ethylene glycol dimethacrylate (EGDMA). Results from (1)H NMR spectroscopy confirmed the covalent incorporation of the peptide into the star periphery. In vitro cytotoxicity experiments showed star polymers to be cytocompatible (≥95% cell viability) and GRGDS-star hybrid hydrogels supported the attachment of MC3T3.E1 (subclone 4) cells. Hybrid hydrogels were prepared by free radical photopolymerization based on 10% (wt/v) PEO dimethacrylates M(n) = 4000 g/mol with 1% (wt/v) GRGDS-star polymers having different peptide content. Cell adhesiveness was also determined from thin film coatings prepared with GRGDS-containing star polymers on nonadherent plastic plates. After 24 h incubation, phase contrast microscopy and scanning electron microscopy (SEM) images showed uniform cell adhesion and distribution over the film containing cell-adhesive star polymers. These results confirm that incorporation of RGD ligand-binding motifs into PEO-based star polymers is required to influence substrate-cell interactions.

Cellular uptake of functional nanogels prepared by inverse miniemulsion ATRP with encapsulated proteins, carbohydrates, and gold nanoparticles.

Atom transfer radical polymerization (ATRP) was used to produce a versatile drug delivery system capable of encapsulating a range of molecules. Inverse miniemulsion ATRP permitted the synthesis of biocompatible and uniformly cross-linked poly(ethylene oxide)-based nanogels entrapping gold nanoparticles, bovine serum albumin, rhodamine B isothiocyanate-dextran, or fluoresceine isothiocyanate-dextran. These moieties were entrapped to validate several biological outcomes and to model delivery of range of molecules. Cellular uptake of nanogels was verified by transmission electron microscopy, gel electrophoresis, Western blotting, confocal microscopy, and flow cytometry. Fluorescent colocalization of nanogels with a fluorophore-conjugated antibody for clathrin indicated clathrin-mediated endocytosis. Furthermore, internalization of nanogels either with or without GRGDS cell attachment-mediating peptides was quantified using flow cytometry. After 45 min of incubation, the uptake of unmodified FITC-Dx-loaded nanogels was 62%, whereas cellular uptake increased to >95% with the same concentration of GRGDS-modified FITC-Dx nanogels. In addition, a spheroidal coculture of human umbilical vascular endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) validated cell endocytosis. Application of ATRP enabled the synthesis of a functionalized drug delivery system with a uniform network that is capable of encapsulating and delivering inorganic, organic, and biological molecules.

Influence of the degree of methacrylation on hyaluronic acid hydrogels properties.

The properties of hyaluronic acid (HA) hydrogels having a broad range of methacrylation are presented. Increasing solubility of glycidyl methacrylate (GM) in a co-solvent mixture during the methacrylation of HA with GM was shown to produce photopolymerizable HAGM conjugates with various degree of methacrylation (DM) ranging from 14% up to 90%. Aqueous solutions of HAGM macromonomers were photocross-linked to yield hydrogels with nearly full vinyl group conversions after 10 min exposure under ultraviolet light (UV). Hydrogels were characterized by uniaxial compression and volumetric swelling measurements. Keeping the DM constant, the shear modulus was varied from 16 kPa up to 73 kPa by varying the macromonomer concentration. However, at a given macromonomer concentration while varying the DM, similarly the shear modulus varied from 22 kPa up to 65 kPa. Preliminary in-vitro cell culture studies showed that GRGDS modified HAGM hydrogels promoted similarly cell interaction at both low and high DMs, 32% and 60%, respectively. Densely cross-linked hydrogels with a high DM have been shown to be more mechanically robust while maintaining cytocompability and cell adhesion.

Synthesis, mechanical properties, biocompatibility, and biodegradation of polyurethane networks from lysine polyisocyanates.

Bone defects, such as compressive fractures in the vertebral bodies, are frequently treated with acrylic bone cements (e.g., PMMA). Although these biomaterials have sufficient mechanical properties for fixing the fracture, they are non-degradable and do not remodel or integrate with host tissue. In an alternative approach, biodegradable polyurethane (PUR) networks have been synthesized that are designed to integrate with host tissue and degrade to non-cytotoxic decomposition products. PUR networks have been prepared by two-component reactive liquid molding of low-viscosity quasi-prepolymers derived from lysine polyisocyanates and poly(epsilon-caprolactone-co-DL-lactide-co-glycolide) triols. The composition, thermal transitions, and mechanical properties of the biomaterials were measured. The values of Young's modulus ranged from 1.20-1.43 GPa, and the compressive yield strength varied from 82 to 111 MPa, which is comparable to the strength of PMMA bone cements. In vitro, the materials underwent controlled biodegradation to non-cytotoxic decomposition products, and supported the attachment and proliferation of MC3T3 cells. When cultured in osteogenic medium on the PUR networks, MC3T3 cells deposited mineralized extracellular matrix, as evidenced by von Kossa staining and tetracycline labeling. Considering the favorable mechanical and biological properties, as well as the low-viscosity of the reactive intermediates used to prepare the PUR networks, these biomaterials are potentially useful as injectable, biodegradable bone cements for fracture healing.

Gene expression profiling of bone marrow stromal cells from juvenile, adult, aged and osteoporotic rats: with an emphasis on osteoporosis.

PURPOSE: Osteoporosis is a multi-factorial, age-related disease with a complex etiology and mode of regulation involving a large numbers of genes. To better understand the possible relationships among genes, we fingerprinted genes in a rat model induced by ovariectomy to determine differences among osteoporotic, non-osteoporotic, aged and juvenile rats. METHODS: We applied genome wide cDNA microarray technology to analyze genes expressed in bone marrow mesenchymal stromal cells (BMSC) and compared non-osteoporotic adult vs. osteoporotic, non-osteoporotic adult vs. aged, and non-osteoporotic adult vs. juvenile. Rigorous statistical analysis of functional annotation (EASE program) identified over-represented biological and molecular functions with significant group wide changes (p< or =0.05). Some of the expressed genes were further confirmed by quantitative RT-PCR (reverse transcription-polymerase chain reaction). RESULTS: Differences in gene expression were observed by identifying transcripts selected by t-test that were consistently changed by a minimum of two-fold. There were 195 transcripts that showed an increased expression and 109 transcripts that showed decreased expression relative to the osteoporotic condition. Of these, 75% transcripts were unknown gene products or ESTs (expressed sequence tag). A number of genes found in the aged and juvenile groups were not present in the osteoporotic rats. Functional clustering of the genes using the EASE bioinformatics program revealed that transcripts in osteoporosis were associated with signal transduction, lipid metabolism, protein metabolism, ionic and protein transport, neuropeptide and G protein signaling pathways. Although some of the genes have previously been shown to play a key role in osteoporosis, several genes were uniquely identified in this study and likely play a role in developing aged related osteoporosis that could have compelling implications in the development of new diagnostic strategies and therapeutics for osteoporosis. CONCLUSIONS: These data suggest that osteoporosis is associated with changes of multiple novel gene expression and that numerous pathways could play important roles in osteoporosis pathogenesis.

Osteoblast differentiation in vitro and in vivo promoted by Osterix.

C3H10T1/2/Osx, a stably transfected cell line with Osterix (Osx), was produced and chondrocytic and osteoblastic differentiation were studied in vitro. Osx promoted osteoblastic lineage that was dexamethasone dependent. Furthermore, in vivo, Osx induced ectopic mineralization in a heterotopic mouse muscle model. Skeletogenesis involves a cascade of molecular activities sequentially performed by osteoblasts and chondroblasts. A transcriptional factor gene Osx appears to influence cell disposition toward the chondrocytic or osteoblastic phenotype and therefore may be an important signaling cue for bone formation. Understanding the molecular conditions involved with Osx promoted osteoblast differentiation will facilitate therapeutic applications of Osx. Consequently, the objective of this study was to investigate chondrocytic and osteoblastic phenotype differentiation using an Osx plasmid DNA exploiting both in vitro and in vivo methodologies. In vitro, a stably transfected C3H10T1/2/Osx cell line was established and promotion of either an osteoblast or chondroblast phenotype was performed by selectively introducing dexamethasone (dex) and assaying mRNA content and phenotype markers. In vivo, a mouse muscle model was used to determine heterotopic ossification using designated Osx plasmid DNA doses incorporated in a (50:50 Poly (D,L-lactide-co-glycolide) (i.e., PLGA) 3D scaffold. Histological assessment was used to determine implant responses. Quantitative real-time polymerase chain reaction (q-RT-PCR) showed a significant increase in mRNA expression of osteocalcin (Ocn), Runx2, osteonectin (On) and osteopontin (Op) (p < 0.05) in the C3H10T1/2/Osx cells compared to the empty vector transfected cell control. At day 21, mineralization was demonstrated in the cultures of C3H10T1/2/Osx exposed to dex, but neither in cultures lacking dex nor controls. In the absence of dex, C3H10T1/2/Osx cells revealed a significantly higher expression of Sox9 and Aggrecan (Agc). In vivo, 80 microg of Osx plasmid DNA induced heterotopic mineralization 4 weeks following implantation in a mouse muscle model and the effect was dependent on the Osx plasmid DNA dose delivered in the PLGA scaffold. Using a non-committed cell line (C3H10T1/2), cell differentiation to an osteoblast phenotype appears to be dependent upon an interaction between intracellular events initiated by the transcriptional factor Osx and the presence of dex. The in vivo findings suggest Osx may promote osteoblast differentiationand mineralization at a heterotopic site.

Synthesis and characterization of segmented poly(esterurethane urea) elastomers for bone tissue engineering.

Segmented polyurethanes have been used extensively in implantable medical devices, but their tunable mechanical properties make them attractive for examining the effect of biomaterial modulus on engineered musculoskeletal tissue development. In this study, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized from 1,4-diisocyanatobutane, a poly(epsilon-caprolactone) (PCL) macrodiol soft segment and a tyramine-1,4-diisocyanatobutane-tyramine chain extender. By systematically increasing the PCL macrodiol molecular weight from 1100 to 2700Da, the storage modulus, crystallinity and melting point of the PCL segment were systematically varied. In particular, the melting temperature, T(m), increased from 21 to 61 degrees C and the storage modulus at 37 degrees C increased from 52 to 278MPa with increasing PCL macrodiol molecular weight, suggesting that the crystallinity of the PCL macrodiol contributed significantly to the mechanical properties of the polymers. Bone marrow stromal cells were cultured on rigid polymer films under osteogenic conditions for up to 21 days. Cell density, alkaline phosphatase activity, and osteopontin and osteocalcin expression were similar among PEUURs and comparable to poly(d,l-lactic-coglycolic acid). This study demonstrates the suitability of this family of PEUURs for tissue engineering applications, and establishes a foundation for determining the effect of biomaterial modulus on bone tissue development.

Fracture healing in the elderly patient.

Clinical experience gives rise to the impression that there are differences in fracture healing in different age groups. It is evident that fractures heal more efficiently in children than in adults. However, minimal objective knowledge exists to evaluate this assumption. Temporal, spatial, and cellular quantitative and qualitative interrelationships, as well as signaling molecules and extracellular matrix have not been comprehensively and adequately elucidated for fracture healing in the geriatric skeleton. The biological basis of fracture healing will provide a context for revealing the pathophysiology of delayed or even impaired bone regeneration in the elderly. We will summarize experimental studies on age-related changes at the cellular and molecular level that will add to the pathophysiological understanding of the compromised bone regeneration capacity believed to exist in the elderly patient. We will suggest why this understanding would be useful for therapeutics focused on bone regeneration, in particular fracture healing at an advanced age.

Synthesis and in vitro biocompatibility of injectable polyurethane foam scaffolds.

The development of therapeutics for orthopedic clinical indications exploiting minimally invasive surgical techniques has substantial benefits, especially for treatment of fragility fractures in the distal radius of osteoporotics and vertebral compression fractures. We have designed six formulations of injectable polyurethane foams to address these clinical indications. The polyurethanes were prepared by mixing two liquid components and injecting the reactive liquid mixture into a mold where it hardens in situ. Porous polyurethane foams were synthesized from lysine methyl ester diisocyanate, a poly(epsilon-caprolactone-co-glycolide) triol, a tertiary amine catalyst, anionic and non-ionic stabilizers, and a fatty acid pore opener. The rise time of the foams varied from 8-20 min. The porosity was approximately 95% and the pores varied in size from 100-1000 microm. The polyurethane foams supported attachment of viable (>95%) MG-63 cells under dynamic seeding conditions. We anticipate compelling opportunities will be available as a consequence of the favorable biological and physical properties of the injectable polyurethane foams.

rhBMP-2 delivered in a calcium phosphate cement accelerates bridging of critical-sized defects in rabbit radii.

BACKGROUND: Treatment of segmental bone loss remains a challenge in skeletal repairs. This study was performed to evaluate the efficacy of the use of recombinant bone morphogenetic protein-2 (rhBMP-2) delivered in an injectable calcium phosphate cement (alpha bone substitute material [alpha-BSM]) to bridge critical-sized defects in the rabbit radius. METHODS: Unilateral 20-mm mid-diaphyseal defects were created in the radii of thirty-six skeletally mature New Zealand White rabbits. The defects in twelve rabbits each were filled with 0.166 mg/mL rhBMP-2/alpha-BSM cement, 0.033 mg/mL rhBMP-2/alpha-BSM cement, or buffer/alpha-BSM cement. Six rabbits from each group were killed at four weeks, and six were killed at eight weeks. Serial radiographs were made to monitor defect-bridging and residual alpha-BSM carrier. A semiquantitative histological scoring system was used to evaluate defect-bridging. Histomorphometry was used to quantify residual alpha-BSM; trabecular bone area; trabecular bone volume fraction; and cortical length, width, and area. RESULTS: At four weeks, there had been more rapid resorption of alpha-BSM and filling of the defects with trabecular bone in the group treated with 0.166 mg/mL rhBMP-2/alpha-BSM than in the other two groups. Histomorphometry confirmed an increased trabecular area and volume fraction in this group compared with the other two groups. In both rhBMP-2/alpha-BSM-treated groups, the majority of the trabecular bone was formed by a direct process adjacent to the resorbing alpha-BSM. At eight weeks, complete cortical bridging and regeneration of the marrow space were present in all of the defects treated with 0.166 mg/mL rhBMP-2/alpha-BSM. That group also had reduced residual alpha-BSM and trabecular area and volume, compared with the other two groups, at eight weeks as a result of a rapid remodeling process. CONCLUSIONS: Treatment of a critical-sized defect in a rabbit radius with 0.166 mg/mL rhBMP-2/alpha-BSM injectable cement can result in bridging with cortical bone and a regenerated bone-marrow space by eight weeks. Site-specific remodeling appears to be responsible for corticalization and marrow regeneration. CLINICAL RELEVANCE: RhBMP-2 delivered in a calcium phosphate cement may be useful to achieve bridging of critical-sized defects in patients. Its injectable properties may allow minimally invasive use. Delayed percutaneous administration would also be possible when augmentation is desired following an initial surgical procedure or when soft-tissue injuries preclude adequate initial treatment.

Bone marrow stromal cells of young and adult rats respond similarly to platelet-released supernatant and bone morphogenetic protein-6 in vitro.

BACKGROUND: Age-related changes in periodontal bone regeneration, osseointegration of dental implants, and graft consolidation are increasingly considered in treatment planning. This study was intended to show whether aging is associated with a diminished responsiveness of osteoprogenitor cells to growth and differentiation factors. METHODS: We compared the capacity of bone marrow stromal cells harvested from young and adult rats to proliferate, migrate, and differentiate into the osteogenic lineage following exposure to platelet-released supernatant (PRS) or bone morphogenetic protein-6 (BMP-6). Bone marrow stromal cells were isolated from 12 young rats aged 6 weeks and 12 adult rats aged 9 months. Proliferation was assessed by 3[H]thymidine incorporation, migration was evaluated with the Boyden chamber assay, and osteogenic differentiation was deduced from alkaline phosphatase activity. RESULTS: Irrespective of the donor age, bone marrow stromal cells showed increased mitogenic activity and chemotactic motility when exposed to PRS. Adult bone marrow stromal cells had higher alkaline phosphatase activities at baseline and upon incubation with BMP-6 than cells obtained from young animals. There was no difference between the two groups in the slope of the alkaline phosphatase activity curve following stimulation with BMP-6. CONCLUSIONS: The data demonstrate that, irrespective of their age, bone marrow stromal cells respond similarly to PRS and BMP-6 under in vitro conditions. These findings suggest that osteoprogenitor cells within the bone marrow of adult rats retain their juvenile potential to respond to growth and differentiation factors, which are released naturally or are applied therapeutically at sites of bone regeneration.