Heterobifunctional poly(ethylene glycol)-tethered bone morphogenetic protein-2-stimulated bone marrow mesenchymal stromal cell differentiation and osteogenesis

We describe a biomimetic mode of insoluble signaling stimulation to provide target delivery of bone morphogenetic protein-2 (BMP-2), with the aim of prolonging the retention of BMP-2 use in bone tissue engineering and to enable its localized release in response to cellular activity. In our novel localization process, we used heterobifunctional acrylate-N-hydroxysuccinimide poly(ethylene glycol) (PEG) as a spacer to tether BMP-2 onto a poly(lactide-co-glycolide) scaffold. Use of PEG-tethered BMP-2 was feasible because BMP-2 retained its activity after covalent conjugation. The PEG-tethered BMP-2 conjugate sustained stimulation and retained its mitogenic activity, notably affecting pluripotent stem cell proliferation and differentiation. We seeded the scaffolds with bone marrow-derived mesenchymal stromal cells as progenitor cells to evaluate their morphology and phenotypic expression. We also created bilateral, full-thickness cranial defects in rabbits to investigate the osteogenic effect of cultured mesenchymal stromal cells on bone regeneration in vivo. Histomorphometry and histology demonstrated that the PEG-tethered BMP-2 conjugate enhanced de novo bone formation after surgery. Our work revealed the potential for biomimetic surface engineering by entrapping signaling growth factor to stimulate osteogenesis. Our technique may provide a new platform for bone-engineered stem cell therapies.     

Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(L-lactic-co-glycolic acid) scaffold

In this study, a heparin-conjugated poly(l-lactic-co-glycolic acid) (HP-PLGA) scaffold was developed for the sustained delivery of bone morphogenetic protein-2 (BMP-2), and then used to address the hypothesis that BMP-2 delivered from this scaffold could enhance ectopic bone formation. We found the amount of heparin conjugated to the PLGA scaffolds could be increased up to 3.2-fold by using scaffolds made from star-shaped PLGA, as compared to scaffolds made from linear PLGA, and that the release of BMP-2 from the HP-PLGA scaffold was sustained for at least 14 days in vitro. The BMP-2 released from the HP-PLGA scaffold stimulated an increase in alkaline phosphatase (ALP) activity of osteoblasts for 14 days in vitro, suggesting that the HP-PLGA scaffold delivery system releases BMP-2 in a bioactive form for a prolonged period. By contrast, BMP-2 release from unmodified (no heparin) PLGA scaffolds induced a transient increase in ALP activity for the first 3 days and a decrease thereafter. In vivo bone formation studies showed the BMP-2-loaded HP-PLGA scaffolds induced bone formation to a much greater extent than did either BMP-2-loaded unmodified PLGA scaffolds or unloaded (no BMP-2) HP-PLGA scaffolds, with 9-fold greater bone formation area and 4-fold greater calcium content in the BMP-2-loaded HP-PLGA scaffold group compared to the BMP-2-loaded unmodified PLGA scaffold group. Collectively, these results demonstrate that the HP-PLGA delivery system is capable of potentiating the osteogenic efficacy of BMP-2, and underscore its importance as a possible bone regeneration strategy.     

Controlled drug release from a novel injectable biodegradable microsphere/scaffold composite based on poly(propylene fumarate)

The ideal biomaterial for the repair of bone defects is expected to have good mechanical properties, be fabricated easily into a desired shape, support cell attachment, allow controlled release of bioactive factors to induce bone formation, and biodegrade into nontoxic products to permit natural bone formation and remodeling. The synthetic polymer poly(propylene fumarate) (PPF) holds great promise as such a biomaterial. In previous work we developed poly(DL-lactic-co-glycolic acid) (PLGA) and PPF microspheres for the controlled delivery of bioactive molecules. This study presents an approach to incorporate these microspheres into an injectable, porous PPF scaffold. Model drug Texas red dextran (TRD) was encapsulated into biodegradable PLGA and PPF microspheres at 2 microg/mg microsphere. Five porous composite formulations were fabricated via a gas foaming technique by combining the injectable PPF paste with the PLGA or PPF microspheres at 100 or 250 mg microsphere per composite formulation, or a control aqueous TRD solution (200 microg per composite). All scaffolds had an interconnected pore network with an average porosity of 64.8 +/- 3.6%. The presence of microspheres in the composite scaffolds was confirmed by scanning electron microscopy and confocal microscopy. The composite scaffolds exhibited a sustained release of the model drug for at least 28 days and had minimal burst release during the initial phase of release, as compared to drug release from microspheres alone. The compressive moduli of the scaffolds were between 2.4 and 26.2 MPa after fabrication, and between 14.9 and 62.8 MPa after 28 days in PBS. The scaffolds containing PPF microspheres exhibited a significantly higher initial compressive modulus than those containing PLGA microspheres. Increasing the amount of microspheres in the composites was found to significantly decrease the initial compressive modulus. The novel injectable PPF-based microsphere/scaffold composites developed in this study are promising to serve as vehicles for controlled drug delivery for bone tissue engineering.     

Orthotopic bone formation by implantation of apatite-coated poly(lactide-co-glycolide)/hydroxyapatite composite particulates and bone morphogenetic protein-2

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. In the present study, we tested the suitability of apatite-coated poly(D,L-lactide-co-glycolide)/nanohydroxyapatite (PLGA/HA) particulates as carriers for the controlled release of BMP-2. The release of BMP-2 from apatite-coated PLGA/HA particulates was sustained for at least 4 weeks in vitro. A delivery system of apatite-coated PLGA/HA particulates suspended in fibrin gel further slowed the BMP-2 release rate. In vivo implantation of either Fibrin gel + BMP-2 or Fibrin gel + apatite-coated PLGA/HA particulates showed enhanced new bone formation in critical-sized calvarial defects of rats 8 weeks after implantation, compared to implantation of fibrin gel only. Importantly, new bone formation was much higher in the defects treated with BMP-2 delivery using apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + PLGA/HA + BMP-2 group) than in the defects treated either with apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + BMP-2 group) or with BMP-2 delivery using fibrin gel alone (Fibrin gel + BMP-2 group). BMP-2 and osteoinductive HA had an additive effect on orthotopic bone formation. In conclusion, the apatite-coated PLGA/HA particulates showed good results as carriers for BMP-2. The BMP-2 delivery system showed high osteogenic capability in a rat calvarial bone defect model. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and would enhance bone regeneration efficacy for the treatment of large bone defects.     

Marrow stromal osteoblast function on a poly(propylene fumarate)/beta-tricalcium phosphate biodegradable orthopaedic composite

The objective of this study was to assess the osteoconductivity of a poly(propylene fumarate)/beta-tricalcium phosphate (PPF/beta-TCP) composite in vitro. We examined whether primary rat marrow stromal cells would attach, proliferate, and express differentiated osteoblastic function when seeded on PPF/beta-TCP substrates. Attachment studies showed that a confluent monolayer of cells had adhered to the substrates within an 8 h time frame for marrow stromal cells seeded at confluent numbers. Proliferation and differentiated function of the cells were then investigated for a period of 4 weeks for an initial seeding density of 42,000 cells/cm2. Rapid proliferation during the first 24 h as determined by 3H-thymidine incorporation was mirrored by an initial rapid increase in total cell number by DNA assay. A lower proliferation rate and a gradual increase in cell number persisted for the remainder of the study, resulting in a final cell number of 128,000 cells/cm2. Differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity and osteocalcin (OC) production throughout the time course. Both markers of osteoblastic differentiation increased significantly over a 4-week period. By day 28, cells grown on PPF/beta-TCP reached a maximal ALP activity of 11 (+/- 1) x 10(-7) micromol/min/cell, while the OC production reached 40 (+/- 1) x 10(-6) ng/cell. These data show that a PPF/beta-TCP composite exhibits in vitro osteoconductivity similar to or better than that of control tissue culture polystyrene.     

The influence of collagen and hyaluronan matrices on the delivery and bioactivity of bone morphogenetic protein-2 and ectopic bone formation

Bone morphogenetic protein-2 (BMP-2) is known to enhance fracture healing when delivered via a bovine collagen sponge. However, collagen rapidly releases BMP-2 with a high burst phase that is followed by a low sustained phase. As a result, supra-physiological doses of BMP-2 are often required to successfully treat bone defects. High BMP-2 dosing can introduce serious side effects that include edema, bone overgrowth, cyst-like bone formation and significant inflammation. As the release behavior of BMP-2 carriers significantly affects the efficacy of fracture healing, we sought to compare the influence of two BMP-2 delivery matrices with contrasting release profiles on BMP-2 bioactivity and ectopic bone formation. We compared a thiol-modified hyaluronan (Glycosil?) hydrogel that exhibits a low burst followed by a sustained release of BMP-2 to a collagen sponge for the delivery of three different doses of BMP-2, the bioactivities of released BMP-2 and ectopic bone formation. Analysis of bone formation by micro-computed tomography revealed that low burst followed by sustained release of BMP-2 from a hyaluronan hydrogel induced up to 456% more bone compared to a BMP-2 dose-matched collagen sponge that has a high burst and sustained release. This study demonstrates that BMP-2 released with a low burst followed by a sustained release of BMP-2 is more desirable for bone formation. This highlights the therapeutic potential of hydrogels, particularly hyaluronan-based, for the delivery of BMP-2 for the treatment of bone defects and may help abrogate the adverse clinical effects associated with high dose growth factor use.     

Effect of a poly(propylene fumarate) foaming cement on the healing of bone defects.

Regeneration of skeletal tissues has been recognized as a new means for reconstruction of skeletal defects. We investigated the feasibility of an injectable and expandable porous implant system for in situ regeneration of bone. Therefore, a composite biodegradable foaming cement based on poly(propylene fumarate) was injected into a critical size defect made in the rat tibia. Animals were divided into two groups comparing the foam in the experimental group against sham-operated animals having a drill hole but no implant in the control group. Eight animals were included in each group. Animals were sacrificed at 1, 3, and 7 weeks postoperatively. Implantation sites were then evaluated with histologic and histomorphometric methods. Results of this study showed that defects did not heal in sham-operated animals. In the experimental group, metaphyseal and cortical defects healed within the first postoperative week by formation of immature woven bone. At the site of the cortical drill hole defect, healing was noted to progress to complete closure by formation of mature bone. Histomorphometry corroborated these findings and showed that metaphyseal bone remodeling peaked at 1 week postoperatively and then decreased as healing of the cortical defect progressed. This suggests that near-complete restoration of the original state of the tibial bone occurred in this animal model supporting the concept of in situ bone regeneration by application of engineered biodegradable porous scaffolds. () ()     

Interleukin-11 acts synergistically with bone morphogenetic protein-2 to accelerate bone formation in a rat ectopic model.

We previously demonstrated that recombinant human interleukin-11 (rHuIL-11) induced osteoblast differentiation of C3H10T1/2 progenitor cells and also acted synergistically with recombinant human bone morphogenetic protein-2 (rHuBMP-2) in performing the same function. In this study, we investigated the effect of rHuIL-11 and rHuBMP-2 on bone formation in a rat ectopic model. When placed in rats, implants consisting of polymer-coated gelatin sponges containing various concentrations of rHuBMP-2 showed a dose-dependent increase in calcium content. This was confirmed by radiographic analysis of the implants. Although implants containing rHuIL-11 alone did not accumulate calcium, implants containing a combination of rHuBMP-2 and rHuIL-11 had significantly higher calcium levels than those containing rHuBMP-2 alone. This increase was rHuIL-11 dose dependent. The synergistic effect of 20 micrograms rHuIL-11 and 6 micrograms rHuBMP-2 on bone formation was estimated to be 1 week in advance of that of 6 micrograms rHuBMP-2 alone. Histologic examination revealed that the combination of rHuIL-11 and rHuBMP-2 caused spindle cells to accumulate around implants and induced cell infiltration into implants. Bone formation occurred faster in implants with the combination of rHuIL-11 and rHuBMP-2 compared with rHuBMP-2 alone. These results suggest that rHuIL-11 acts synergistically with rHuBMP-2 to more rapidly stimulate bone formation compared with rHuBMP-2 alone. This novel combined therapy may be of great clinical benefit in bone healing.     

Degradation and biocompatibility of a poly(propylene fumarate)-based/alumoxane nanocomposite for bone tissue engineering

In this work, we evaluated the in vitro cytotoxicity and in vivo biocompatibility of a novel poly(propylene fumarate) (PPF)-based/alumoxane nanocomposite for bone tissue engineering applications. The incorporation of functionalized alumoxane nanoparticles into the PPF-based polymer was previously shown to significantly increase the material's flexural mechanical properties. In the current study, samples underwent accelerated in vitro degradation to allow the study of biological responses to these materials over the course of their degradation on a shortened timescale. The polymer, a macrocomposite composed of the polymer and micron-sized particles, and the nanocomposite were evaluated at three stages of degradation for in vitro cytotoxicity with a fibroblast cell line and in vivo soft-tissue response after 3 and 12 weeks of implantation in adult goats. All three material groups experienced mass loss during degradation, but the nanocomposite group eroded significantly faster than the other groups. Nondegraded materials demonstrated minimal cytotoxicity and a minor inflammatory response in soft tissue. On the contrary, predegraded samples elicited more pronounced responses, though these were due to the increase in surface area, surface roughness, and fragmentation associated with the degradation process. The presence of alumoxane nanoparticles in the PPF-based nanocomposite did not significantly affect its cytotoxicity or biocompatibility.     

Photocrosslinking characteristics and mechanical properties of diethyl fumarate/poly(propylene fumarate) biomaterials

The development of tissue engineered materials for the treatment of large bone defects would provide attractive alternatives to the autografts, allografts, non-degradable polymers, ceramics, and metals that are currently used in clinical settings. To this end, poly(propylene fumarate) (PPF), a viscous polyester synthesized from diethyl fumarate (DEF), has been studied for use as an engineered bone graft. We have investigated the photocrosslinking of PPF dissolved in its precursor, DEF, using the photoinitiator bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO) and low levels of ultraviolet light exposure. A three factor, 2 x 2 x 4 factorial design was developed, studying the effects of PPF number average molecular weight, BAPO initiator content, and DEF content upon photocrosslinking characteristics and mechanical properties. Uncured DEF/PPF solution viscosity fell over three orders of magnitude as DEF content was increased from 0% to 75%. The exothermic photocrosslinking reaction released low levels of heat, with no more than 160J/g released from any formulation tested. As a result, the maximum photocrosslinking temperature remained below 47 degrees C for all samples. Both sol fraction and swelling degree generally increased with increasing DEF content. Compressive mechanical properties were within the range of trabecular bone, with the strongest samples possessing an elastic modulus of 195.3 +/- 17.5 MPa and a fracture strength of 68.8 +/- 9.4MPa. Finally, the results indicate that PPF crosslinking was facilitated at low DEF precursor concentrations, but hindered at higher precursor concentrations. These novel DEF/PPF solutions may be preferred over pure PPF as the basis for an engineered bone graft because they (1) exhibit reduced viscosity and thus are easily handled, (2) form polymer networks with compressive strength at fracture suitable for consideration for trabecular bone replacement, and (3) may be readily fabricated into solids with a wide range of structures.     

Quantitative measures of osteoinductivity of a porous poly(propylene fumarate) bone graft extender

Bioresorbable bone graft substitutes could alleviate disadvantages associated with the use of autografts, allografts, and other synthetic materials. However, little is known about the minimum autograft/extender ratio for a given material at which a sufficient osteoinductive effect is still seen. Therefore, we investigated a bioresorbable bone graft substitute made from the unsaturated polyester poly(propylene fumarate), PPF, at various mixing ratios with autograft. The bone graft extender is cross-linked in the presence of a hydroxylapatite filler and effervescent foaming agents citric acid and sodium bicarbonate. The porous bone graft extender material develops porosity in vivo by generating carbon dioxide during the effervescent reaction, resulting in foam formation and expansion with respective pore sizes of 50 to 1000 microm. In an attempt to determine how much cancellous autograft bone could be extended with the poly(propylene fumarate) material and at which ratio the autograft/extender combination remained supportive of the overall structural integrity of the repairing defect site, we studied the amount of new bone formation on implantation of the materials in 3-mm holes made in the anteromedial tibial metaphysis of Sprague-Dawley rats. The extender formulation was analyzed at high autograft/extender (75% autograft/25% extender) and low autograft/extender (25% autograft/75% extender) mixing ratios and compared with negative (extender alone) and positive (autograft alone) controls. Animals from each of the formulations were killed in groups of eight at 6 weeks postoperatively. Hence, a total of 32 animals were included in this study. Histologic analysis of the healing process revealed enhanced in vivo osteoinduction with the bone graft extender regardless of the autograft loading. Histomorphometry did not show any statistically significant difference between the high and low autograft/extender ratios. All formulations maintained implant integrity and did not provoke sustained inflammatory responses. This study suggested that the presence of even a small amount of autograft within the polymer-based bone graft extender results in significant enhancement of osteoinduction. This finding has immediate applicability to the development of bone graft extender formulations for clinical use.     

Injectable, in situ forming poly(propylene fumarate)-based ocular drug delivery systems

This study sought to develop an injectable formulation for long-term ocular delivery of fluocinolone acetonide (FA) by dissolving the anti-inflammatory drug and the biodegradable polymer poly(propylene fumarate) (PPF) in the biocompatible, water-miscible, organic solvent N-methyl-2-pyrrolidone (NMP). Upon injection of the solution into an aqueous environment, a FA-loaded PPF matrix is precipitated in situ through the diffusion/extraction of NMP into surrounding aqueous fluids. Fabrication of the matrices and in vitro release studies were performed in phosphate buffered saline at 37 degrees C. Drug loadings up to 5% were achieved. High performance liquid chromatography was employed to determine the released amount of FA. The effects of drug loading, PPF content of the injectable formulation, and additional photo-crosslinking of the matrix surface were investigated. Overall, FA release was sustained in vitro over up to 400 days. After an initial burst release of 22 to 68% of initial FA loading, controlled drug release driven by diffusion and bulk erosion was observed. Drug release rates in a therapeutic range were demonstrated. Release kinetics were found to be dependent on drug loading, formulation PPF content, and extent of surface crosslinking. The results suggest that injectable, in situ formed PPF matrices are promising candidates for the formulation of long-term, controlled delivery devices for intraocular drug delivery.     

Experimental studies on bone induction using low-molecular-weight poly (DL-lactide-co-glycolide) as a carrier for recombinant human bone morphogenetic protein-2

An appropriate carrier acting as a slow delivery vehicle for the BMPs is required for maximal clinical effectiveness of these bone-inductive proteins. The purpose of this study was to evaluate a low-molecular-weight PLGA copolymer as a synthetic, biodegradable carrier for rhBMP-2 implantation in vivo. Two, 10, or 50 microg of recombinant human BMP-2 were mixed with 10 mg of a poly (DL-lactide-co-glycolide) (PLGA) 50:50 copolymer and implanted into the calf muscles of Wistar rats. Soft X-ray analysis and histologic examination indicated that new bone formation occurred at all rhBMP-2-implanted sites within 3 weeks after implantation. Correlation of rhBMP-2 concentration with the amount of bone induction was confirmed by specific alkaline phosphatase activity and calcium content assay. In vitro analysis indicated that 78.5% of the PLGA copolymer was degraded to smaller molecular weight material after 14 days in PBS solution. It is suggested that rhBMP-2 was released in an active form at the implant site during the degradation of the copolymer, resulting in the induction of new bone formation. Thus this low-molecular-weight PLGA copolymer material represents a promising delivery vehicle for BMPs, and possibly other growth factors, around dental and orthopedic implants.     

Potentiation of the activity of bone morphogenetic protein-2 in bone regeneration by a PLA-PEG/hydroxyapatite composite

Bone morphogenetic proteins (BMPs) are biologically active molecules capable of inducing new bone formation, and show potential for clinical use in bone defect repair. However, an ideal system for delivering BMPs that can potentiate their bone-inducing ability and provide initial mechanical strength and scaffold for bone ingrowth has not yet been developed. In this study, to construct a carrier/scaffold system for BMPs, we combined two biomaterials: interconnected-porous calcium hydroxyapatite ceramics (IP-CHA), and the synthetic biodegradable polymer poly D,L,-lactic acid-polyethyleneglycol block co-polymer (PLA-PEG). We used a rabbit radii model to evaluate the bone-regenerating efficacy of rhBMP-2/PLA-PEG/IP-CHA composite. At 8 weeks after implantation, all bone defects in groups treated with 5 or 20 microg of BMP were completely repaired with sufficient strength. Furthermore, using this carrier scaffold system, we reduced the amount of BMP necessary for such results to about a tenth of the amount needed in previous studies, probably due to the superior osteoconduction ability of IP-CHA and the optimal drug delivery system provided by PLA-PEG, inducing new bone formation in the interconnected pores. The present findings indicate that the synthetic biodegradable polymer/IP-CHA composite is an excellent combination carrier/scaffold delivery system for rhBMP-2, and that it strongly promotes the clinical effects of rhBMP-2 in bone tissue regeneration.     

Enhanced bioactivity of a poly(propylene fumarate) bone graft substitute by augmentation with nano-hydroxyapatite

The bioactivity of a nano-hydroxyapatite-augmented, bioresorbable bone graft substitute made from the unsaturated polyester, poly(propylene fumarate), was analyzed by evaluating biocompatibility and osteointegration of implants placed into a rat tibial defect. Three groups of eight animals each were evaluated by grouting bone graft substitutes into 3-mm holes that were made into the anteromedial tibial metaphysis of rats. Thus, a total of 24 animals was included in this study. Two different formulations varying as to the type of hydroxyapatite were used: Group 1 - nano-hydroxyapatite, Group 2 - micron-hydroxyapatite, with a Group 3 control defect remaining unfilled. Animals of each of the three groups were sacrificed in groups of eight at postoperative week three. Histologic analysis revealed best superior biocompatibility and osteointegration of bone graft substitutes when nanohydroxyapatite was employed. At three weeks, there was more reactive new bone formation in this group when compared to the micron-hydroxyapatite group. The control group showed incomplete closure of the defect. This study suggested that nano-hydroxyapatite may improve upon the bioactivity of bone implant and repair materials. The model scaffold used in this study, poly(propylene fumarate), appeared to provide an osteoconductive pathway by which bone will grow in faster. Clinical implications of the use potential advantages of nano-hydroxyapatite on bone repair and orthopaedic implant design are discussed.     

Effects of transforming growth factor beta1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates

Recombinant human transforming growth factor beta1 (TGF-beta1) was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) to create a delivery vehicle for the growth factor. The entrapment efficiency of TGF-beta1 in the microparticles containing 5% PEG was 40.3 +/- 1.2% for a TGF-beta1 loading density of 6.0 ng/1 mg of microparticles. For the same loading, 17.9 +/- 0.6 and 32.1 +/- 2.5% of the loaded TGF-beta1 was released after 1 and 8 days, respectively, followed by a plateau for the remaining 3 weeks. Rat marrow stromal cells showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating the activity of TGF-beta1 was retained during microparticle fabrication and after TGF-beta1 release. An optimal TGF-beta1 dosage of 1.0 ng/mL was determined through a 3-day dose response study for maximal alkaline phosphatase (ALP) activity. The TGF-beta1 released from the microparticles loaded with 6.0 ng TGF-beta1/1 mg of microparticles for the optimal dosage of TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells cultured on poly(propylene fumarate) substrates. The cells showed significantly increased total cell number, ALP activity, and osteocalcin production with values reaching 138,700 +/- 3300 cells/cm(2), 22.8 +/- 1.5 x 10(-7) micromol/min/cell, and 15.9 +/- 1.5 x 10(-6) ng/cell, respectively, after 21 days as compared to cells cultured under control conditions without TGF-beta1. These results suggest that controlled release of TGF-beta1 from the PLGA/PEG blend microparticles may find applications in modulating cellular response during bone healing at a skeletal defect site.     

Tissue-engineered cartilage constructs using composite hyaluronic acid/collagen I hydrogels and designed poly(propylene fumarate) scaffolds

Our approach to cartilage tissue-engineering scaffolds combines image-based design and solid free-form (SFF) fabrication to create load-bearing constructs with user-defined parameters. In this study, 3-dimensional scaffolds with cubic and ellipsoidal pore architecture were fabricated using poly(propylene fumarate) (PPF). To increase seeding efficiency and cellular retention, hydrogels were used to deliver cells into the scaffolds. The first objective of this study was to evaluate the concentrations of composite hyaluronic acid (HyA) and collagen I hydrogels best able to stimulate proteoglycan synthesis in porcine chondrocytes in vitro and in vivo. The second objective was to evaluate the differences in extracellular matrix production due to pore geometry and scaffold design. For the in vitro assessment, chondrocytes were encapsulated in collagen I hydrogels with varying concentrations of HyA. Hydrogels were cultured for 1 and 2 weeks, and then the sulfated glycosaminoglycan (sGAG) content was quantified using a dimethyl-methylene blue assay. The concentration of HyA best able to increase ECM synthesis was 5% HyA/collagen I, or 0.23 mg/mL HyA. The results from the in vitro experiment were used as culture parameters for the in vivo analysis. Composite 5% HyA/collagen I or collagen I-only hydrogels were used to seed chondrocytes into SFF-fabricated scaffolds made of PPF with designed cubic or ellipsoidal pore geometry. The scaffolds were implanted subcutaneously in immunocompromised mice for 4 weeks. Histomorphometric analyses of sections stained with Safranin O were used to quantify the amount of ECM deposited by cells in the scaffolds. Scaffolds seeded with 5% HyA/collagen hydrogels had significantly greater areas of positive Safranin O staining (approximately 60%, compared with 30% for scaffolds with collagen I hydrogels only), indicating that greater numbers of chondrocytes retained their metabolic activity in the ectopic environment. These scaffolds also had greater stain intensities (corresponding to greater amounts of sGAG in the ECM) than their counterparts seeded with collagen I hydrogels alone. Significant differences in matrix production were not found between the scaffold pore designs. Overall, these results indicate that a combination of composite HyA hydrogels and designed SFF scaffolds could provide a functional tissue-engineered construct for cartilage repair with enhanced tissue regeneration in a load-bearing scaffold.     

Ectopic bone formation in mice associated with a lactic acid/dioxanone/ethylene glycol copolymer-tricalcium phosphate composite with added recombinant human bone morphogenetic protein-2

A new putty-like material with bone-inducing capacity was made by combining a block copolymer of poly d,l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) and beta-tricalcium phosphate (beta-TCP) powder with added recombinant human bone morphogenetic protein-2 (rhBMP-2). To optimize the material's efficacy for bone formation, we formulated the optimal composition ratio of the respective constituent that gives the greatest osteoinductive efficacy in a mouse model of ectopic bone formation. In this series of studies, we investigated the size of ectopic bone mass induced 3 and 6 weeks after implantation of the materials composed of 30 mg of PLA-DX-PEG with 2 microg of rhBMP-2 and 0, 15, 30, or 60 mg of beta-TCP powder. An additional experiment was designed to investigate how content ratios of beta-TCP powder in 30 mg-putty implants (0%, 16.7%, 33.3%, 50%, 66.7%, 83.3%, or 100%) for a fixed dose (5 microg) of the rhBMP-2 altered the size of the induced ossicle. The results from the first experiment indicated that the bone yields were linearly dependent on the amount of additional beta-TCP powder. In the second experiment, the largest ossicles induced by 5 microg of rhBMP-2 were obtained when the polymer/beta-TCP ratio was 1/2 in mice. The data provide important insights into the fabrication of implants that provide efficacious delivery of rhBMP-2. The new putty-like material may be valuable for repairing or regenerating bone in a clinical setting.     

壳聚糖-磷酸钙/骨形态发生蛋白2复合骨髓间充质干细胞促进兔脊柱的融合

背景：前期实验已经证明壳聚糖-磷酸钙/骨形态发生蛋白2复合材料能够促进兔脊柱融合。 目的：评价壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子支架材料在兔椎间融合中的应用效果。 方法：制备壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子支架材料,并与大鼠骨髓间充质干细胞在体外构成组织工程骨。摘除40只新西兰大白兔椎间盘,随机分为4组：空白对照组未植入任何材料,对照组植入自体髂骨,支架材料组植入壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子复合材料,实验组植入组织工程骨材料。 结果与结论：术后12周：①X射线片：对照组与实验组椎体融合,两组间融合节段生物力学强度大致相同,生物力学强度高于空白对照组与支架材料组(P < 0.05),且支架材料组高于空白对照组(P < 0.05)。②组织学切片：实验组与对照组有编织骨岛和新生毛细血管生成,支架材料组仅观察到壳聚糖支架网络,空白对照组未发现特殊组织结构。表明壳聚糖-磷酸钙/骨形态发生蛋白2/碱性成纤维细胞生长因子复合鼠骨髓间充质干细胞能够明显促进脊柱融合。     

Recombinant human bone morphogenetic protein-2/atelocollagen composite as a new material for ossicular reconstruction

Ossicular reconstruction is the rebuilding of the damaged middle ear. There are many different prosthesis and techniques used to reconstruct the middle ear ossicles. However, precision in the surgical procedures and prostheses used for ossiculoplasty are still imperfect. The objective of this study was to evaluate the potential of recombinant human bone morphogenetic protein-2 (rhBMP-2)/ atelocollagen composite for ossicular reconstruction implanted in the tympanic cavity of rat. The ossicles were extirpated by perforating the tympanic membranes of rats. rhBMP-2/atelocollagen composite was implanted as substitute of ossicles in intimate contact with the tympanic membrane. Composites were subjected to histological, immunohistochemical, and radiological examination. To evaluate the auditory function, auditory brainstem response (ABR) was measured. rhBMP-2/atelocollagen composites showed good stability and durability without any inflammatory reaction within the tympanic cavity. The process of new bone formation was similar to intramembranous ossification. They also demonstrated that the hearing ability was re-established by ABR threshold shifts. rhBMP-2/atelocollagen composite exhibited excellent potential for ossicular reconstruction, maintaining their vibratory function. This ossicular tissue engineering may be considered as a future therapeutic strategy for ossiculoplasty.