Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the Osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKOOCN mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKOOCN mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKOOCN mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKOOCN mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging.
Histone deacetylase; Osteocalcin-Cre; Osteoblast; Osteocyte; DNA damage
This project was designed to test the hypothesis that rapid intraoperative processing of bone marrow based on hyaluronan (HA) could be used to improve the outcome of local bone regeneration if the concentration and prevalence of marrow-derived connective tissue progenitors (CTPs) could be increased and nonprogenitors depleted before implantation.
HA was used as a marker for positive selection of marrow-derived CTPs using magnetic separation (MS) to obtain a population of HA-positive cells with an increased CTP prevalence. Mineralized cancellous allograft (MCA) was used as an osteoconductive carrier scaffold for loading of HA-positive cells. The canine femoral multidefect model was used and four cylindrical defects measuring 10 mm in diameter and 15 mm in length were grafted with MCA combined with unprocessed marrow or with MS processed marrow that was enriched in HA+ CTPs and depleted in red blood cells and nonprogenitors. Outcome was assessed at 4 weeks using quantitative 3D microcomputed tomography (micro-CT) analysis of bone formation and histomorphological assessment.
Histomorphological assessment showed a significant increase in new bone formation and in the vascular sinus area in the MS-processed defects. Robust bone formation was found throughout the defect area in both groups (defects grafted with unprocessed marrow or with MS processed marrow.) Percent bone volume in the defects, as assessed by micro-CT, was greater in defects engrafted with MS processed cells, but the difference was not statistically significant.
Rapid intraoperative MS processing to enrich CTPs based on HA as a surface marker can be used to increase the concentration and prevalence of CTPs. MCA grafts supplemented with heparinized bone marrow or MS processed cells resulted in a robust and advanced stage of bone regeneration at 4 weeks. A greater new bone formation and vascular sinus area was found in defects grafted with MS processed cells. These data suggest that MS processing may be used to enhance the performance of marrow-derived CTPs in clinical bone regeneration procedures. Further assessment in a more stringent bone defect model is proposed.
Research on biomaterial nerve scaffolds has been carried out for 50 years. Only three materials (collagen, polycaprolactone and polyglycollic acid) have progressed to clinical use. Pre-clinical animal models are critical for testing nerve scaffolds prior to implementation in clinical practice. We have conducted a systematic review of 416 reports in which animal models were used for evaluation of nerve regeneration into synthetic conduits. A valid animal model of nerve regeneration requires it to reproduce the specific processes that take place in regeneration after human peripheral nerve injury. No distinct animal species meets all the requirements for an ideal animal model. Certain models are well suited for understanding regenerative neurobiology while others are better for pre-clinical evaluation of efficacy. The review identified that more than 70 synthetic materials were tested in eight species using 17 different nerves. Nerve gaps ranged from 1 to 90 mm. More than 20 types of assessment methodology were used with no standardization of methods between any of the publications. The review emphasizes the urgent need for standardization or rationalization of animal models and evaluation methods for studying nerve repair.
Peripheral nerve injury; peripheral nerve repair; nerve tube; nerve scaffold; biodegradable
Poly(ε-caprolactone fumarate) (PCLF) scaffold formulations were assessed as a delivery system of recombinant human bone morphogenetic protein (rhBMP-2) for bone tissue engineering. The formulations included PCLF with combinations of poly(vinyl alcohol) (PVA) and hydroxyapatite (HA). The assessments included in vitro and in vivo assays. In vitro assays validated cell attachment using a pre-osteoblast cell line (MC3T3-E1). Additionally, in vitro release profiles of rhBMP-2 from PCLF scaffolds were determined up to 21 days. Data suggested PCLF incorporated with PVA and HA accelerated rhBMP-2 release and the released protein was bioactive. For the in vivo study, a critical sized defect (CSD) model in a rabbit calvaria was used to test PCLF scaffolds. At 6 weeks post-implantation, significantly more bone formation was measured in PCLF scaffolds containing rhBMP-2 than in scaffolds without rhBMP-2. In conclusion, we demonstrated PCLF delivered biologically active rhBMP-2, promoted bone healing in a CSD and has potential as a bone tissue engineering scaffold.
poly(ε-caprolactone fumarate); three-dimensional scaffold; rabbit calvarial critical sized defect; rhBMP-2; bone tissue engineering
Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgical resection and adjunctive chemotherapy are the only widely available options of treatment for this disease. Anti-tumor compound 2-Methoxyestradiol (2-ME) triggers cell death through the induction of apoptosis in osteosarcoma cells, but not in normal osteoblasts. In this report, we have investigated whether autophagy plays a role in 2-ME actions on osteosarcoma cells. Transmission electron microscopy imaging shows that 2-ME treatment leads to the accumulation of autophagosomes in human osteosarcoma cells. 2-ME induces the conversion of the microtubule-associated protein LC3-I to LC3-II, a biochemical marker of autophagy that is correlated with the formation of autophagosomes. Conversion to LC3-II is accompanied by protein degradation in 2-ME-treated cells. 2-ME does not induce autophagosome formation in normal primary human osteoblasts. In addition, 2-ME-dependent autophagosome formation in osteosarcoma cells requires ATG7 expression. Furthermore, 2-ME does not induce accumulation of autophagosomes in osteosarcoma cells that express dominant negative mutant RNA-dependent protein kinase (PKR) and are resistant to anti-proliferative and anti-tumor effects of 2-ME. Taken together, our study shows that 2-ME treatment induces PKR-dependent autophagy in osteosarcoma cells, and that autophagy could play an important role in 2-ME-mediated anti-tumor actions and in the control of osteosarcoma.
Polycaprolactone fumarate (PCLF) is a cross-linkable derivate of polycaprolactone diol that has been shown to be an effective nerve conduit material that supports regeneration across segmental nerve defects and has warranted future clinical trials. Degradation of the previously studied PCLF (PCLFDEG) releases toxic small molecules of diethylene glycol used as the initiator for the synthesis of polycaprolactone diol. In an effort to eliminate this toxic degradation product we present a strategy for the synthesis of PCLF from either propylene glycol (PCLFPPD) or glycerol (PCLFGLY). PCLFPPD is linear and resembles the previously studied PCLFDEG, while PCLFGLY is branched and exhibits dramatically different material properties. The synthesis and characterization of their thermal, rheological, and mechanical properties are reported. The results show that the linear PCLFPPD has material properties similar to the previously studied PCLFDEG. The branched PCLFGLY exhibits dramatically lower crystalline properties resulting in lower rheological and mechanical moduli, and is therefore a more compliant material. In addition, the question of an appropriate FDA approvable sterilization method is addressed. This study shows that autoclave sterilization on PCLF materials is an acceptable sterilization method for cross-linked PCLF and has minimal effect on the PCLF thermal and mechanical properties.
Polycaprolactone fumarate; polyester; sterilization; nerve regeneration
Development of novel therapeutic approaches to repair fracture non-unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non-union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM-MSCs) or a no cell control group (n = 10 to 12 per group). Preliminary studies demonstrated that both for hESC-derived MSCs and hBM-MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque and stiffness were significantly greater in the hBM-MSC as compared to the control group that received no cells; values for these parameters in the hESC-derived MSC group were intermediate between the hBM-MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X-ray in the hESC-derived MSC group. Our results thus indicate that while hESC-derived MSCs may have potential to induce fracture healing in non-unions, hBM-MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells.
embryonic stem cells; mesenchymal stem/stromal cells; osteogenic; bone repair; fracture non-union
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
Accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons.
Simultaneous tracing of the tibial and peroneal nerves with fast blue (FB) and diamidino yellow (DY), 8 weeks after repair of a 1-cm nerve gap in the rat sciatic nerve, was performed to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with DY 1 week before and FB 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons.
In the cases in which there was successful regeneration across single lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair.
Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis in the development of a nerve tube for motor nerve repair.
misdirection; axon targeting; double labeling; peripheral nerve regeneration; rat sciatic nerve model; retrograde tracing
Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.
Osteogenesis imperfecta; C-propeptide; collagen; C-proteinase; mineralization; high bone mass
This study describes the use of oligo [(polyethylene glycol) fumarate] (OPF) hydrogel scaffolds as vehicles for sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the transected spinal cord. dbcAMP was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within the scaffolds architecture. Functionality of the released dbcAMP was assessed using neurite outgrowth assays in PC12 cells and by delivery to the transected spinal cord within OPF seven channel scaffolds, which had been loaded with Schwann cells or mesenchymal stem cells (MSCs). Our results showed that encapsulation of dbcAMP in microspheres lead to prolonged release and continued functionality in vitro. These microspheres were then successfully incorporated into OPF scaffolds and implanted in the transected thoracic spinal cord. Sustained delivery of dbcAMP inhibited axonal regeneration in the presence of Schwann cells but rescued MSC-induced inhibition of axonal regeneration. dbcAMP was also shown to reduce capillary formation in the presence of MSCs, which was coupled with significant functional improvements. Our findings demonstrate the feasibility of incorporating PLGA microsphere technology for spinal cord transection studies. It represents a novel sustained delivery mechanism within the transected spinal cord and provides a platform for potential delivery of other therapeutic agents.
In this study, we have compared the effects of negative and positive fixed charge on chondrocyte behavior in vitro. Electrical charges have been incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) using small charged monomers such as sodium methacrylate (SMA) and (2-(methacryloyloxy) ethyl)-trimethyl ammonium chloride (MAETAC) to produce negatively and positively charged hydrogels, respectively. The hydrogel physical and electrical properties were characterized through measuring and calculating the swelling ratio and zeta potential, respectively. Our results revealed that the properties of these OPF modified hydrogels varied according to the concentration of charged monomers. Zeta potential measurements demonstrated that the electrical property of the OPF hydrogel surfaces changed due to incorporation of SMA and MAETAC and that this change in electrical property was dose-dependent. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy was used to determine the hydrogel surface composition. To assess the effects of surface properties on chondrocyte behavior, primary chondrocytes isolated from rabbit ears were seeded as a monolayer on top of the hydrogels. We demonstrated that the cells remained viable over 7 days and began to proliferate while seeded on top of the hydrogels. Collagen type II staining was positive in all samples; however, the intensity of the stain was higher on negatively charged hydrogels. Similarly, GAG production was significantly higher on negatively charged hydrogels compared to neutral hydrogel. Reverse transcription polymerase chain reaction showed up-regulation of collagen type II and down-regulation of collagen type I on the negatively charged hydrogels. These findings indicate that charge plays an important role in establishing an appropriate environment for chondrocytes and hence in the engineering of cartilage. Thus, further investigation into charged hydrogels for cartilage tissue engineering is merited.
hydrogel; cartilage tissue engineering; OPF; scaffold
Osteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells.
2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α).
2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls.
2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment strategies in the control of osteosarcoma.
2-Methoxyestradiol; osteosarcoma; Interferon; ISRE; GAS
Mechanical and electrical properties of polycaprolactone fumarate-polypyrrole (PCLF-PPy) scaffolds were studied under physiological conditions to evaluate their ability to maintain material properties necessary for application as conductive nerve conduits. PC12 cells cultured on PCLF-PPy scaffolds were stimulated with regimens of 10 μA of constant or 20 Hz frequency current passed through the scaffolds for 1 h/day. PC12 cellular morphologies were analyzed by fluorescence microscopy after 48 h. PCLF-PPy scaffolds exhibited excellent mechanical properties at 37°C which would allow suturing and flexibility. The surface resistivity of the scaffolds was 2kΩ and the scaffolds were electrically stable during application of electrical stimulation (ES). In vitro studies showed significant increases in percentage of neurite bearing cells, number of neurites per cell and neurite length in the presence of ES compared to no ES. Additionally, extending neurites were observed to align in the direction of the applied current. This study shows that electrically conductive PCLF-PPy scaffolds possess material properties necessary for application as nerve conduits. Additionally, the capability to significantly enhance and direct neurite extension by passing electrical current through PCLF-PPy scaffolds renders them even more promising as future therapeutic treatments for severe nerve injuries.
Electrical Stimulation; Polypyrrole; Nerve; PCLF; PC12 cells
In this work, a series of copolymers of polypropylene fumarate-co-polycaprolactone (PPF-co-PCL) were synthesized via a three-step polycondensation reaction of oligomeric polypropylene fumarate (PPF) with polycaprolactone (PCL). The effects of PPF precursor molecular weight, PCL precursor molecular weight, and PCL fraction in the copolymer (PCL feed ratio) on the maximum crosslinking temperature, gelation time, and mechanical properties of the crosslinked copolymers were investigated. The maximum crosslinking temperature fell between 38.2±0.3 and 47.2±0.4 °C, which increased with increasing PCL precursor molecular weight. The gelation time was between 4.2±0.2 and 8.5±0.7 min, and decreased with increasing PCL precursor molecular weight. The compressive moduli ranged from 44±1.8 to 142±7.4 MPa, with enhanced moduli at higher PPF precursor molecular weight and lower PCL feed ratio. The compressive toughness was in the range of 4.1±0.3 and 17.1±1.3 KJ/m3. Our data suggest that the crosslinking and mechanical properties of PPF-co-PCL can be modulated by varying the composition. Therefore the PPF-co-PCL copolymers may offer increased versatility as an injectable, in situ polymerizable biomaterial than the individual polymers of PPF and PCL.
Polypropylene fumarate; polycaprolactone; injectable biomaterials; in situ polymerizable
Regeneration of peripheral nerves after injury is suboptimal. We now report the long term delivery of nerve growth factor (NGF) by biodegradable poly-lactic-co-glycolic acid (PLGA) microspheres in vitro and in vivo. Lactic to glycolic acid ratios of 50:50 and 85:15 were fabricated using the double emulsion solvent, evaporation technique. Three different inherent viscosities (0.1dL/g: 1A, 0.4dL/g: 4A, 0.7dL/g: 7A) were analyzed. In vitro, release of NGF for 23 days was measured. Electron microscopy demonstrated intact spheres for at least 7 days (50:50 1A), 14 days (50:50 4A) or 35 days (50:50 7A and 85:15 7A). In vitro release kinetics were characterized by burst release, followed by release of NGF at a rate of 0.6%-1.6% a day. Release curves for 50:50 1A and 85:15 7A differed significantly from other compositions (p<0.01). In vivo, release was characterized by a novel radionuclide tracking assay. Release rates varied from 0.9%-2.2% per day with linear kinetics. All but the 85:15 type of spheres showed different release profiles in vivo compared to in vitro conditions. Based on the surface morphology and release profiles we found microspheres fabricated from 50:50 4A PLGA to be best suited for the use in a rat sciatic nerve injury model.
Nerve Growth Factor; Microspheres; Peripheral Nerve; Poly-lactic-co-glycolic-acid; Dorsal root ganglia
Bone morphogenetic proteins (BMPs) play a central role in local bone regeneration strategies, whereas the anabolic features of parathyroid hormone (PTH) are particularly appealing for the systemic treatment of generalized bone loss. The aim of the current study was to investigate whether local BMP-2-induced bone regeneration could be enhanced by systemic administration of PTH (1–34). Empty or BMP-2-loaded poly(lactic-co glycolic acid)/poly(propylene fumarate)/gelatin composites were implanted subcutaneously and in femoral defects in rats (n = 9). For the orthotopic site, empty defects were also tested. Each of the conditions was investigated in combination with daily administered subcutaneous PTH (1–34) injections in the neck. After 8 weeks of implantation, bone mineral density (BMD) and bone volume were analyzed using microcomputed tomography and histology. Ectopic bone formation and almost complete healing of the femoral defect were only seen in rats that received BMP-2-loaded composites. Additional treatment of the rats with PTH (1–34) resulted in significantly (p < 0.05) enhanced BMD and bone volume in the BMP-2 composites at both implantation sites. Despite its effect on BMD in the humerus and vertebra, PTH (1–34) treatment had no significant effect on BMD and bone volume in the empty femoral defects and the ectopically or orthotopically implanted empty composites. Histological analysis showed that the newly formed bone had a normal woven and trabecular appearance. Overall, this study suggests that intermittent administration of a low PTH dose alone has limited potential to enhance local bone regeneration in a critical-sized defect in rats. However, when combined with local BMP-2-releasing scaffolds, PTH administration significantly enhanced osteogenesis in both ectopic and orthotopic sites.
The goal of this study was to develop a polymeric carrier for delivery of anti-tumor drugs and sustained release of these agents in order to optimize anti-tumor activity while minimizing systemic effects. We used oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels modified with small negatively charged molecules, sodium methacrylate (SMA), for delivery of doxorubicin (DOX). SMA at different concentrations was incorporated into the OPF hydrogel with a photo-crosslinking method. The resulting hydrogels exhibited sensitivity to the pH and ionic strength of the surrounding environment. Our results revealed that DOX was bound to the negatively charged hydrogel through electrostatic interaction and was released in a timely fashion with an ion exchange mechanism. Release kinetics of DOX was directly correlated to the concentration of SMA in the hydrogel formulations. Anti-tumor activity of the released DOX was assessed using a human osteosarcoma cell line. Our data revealed that DOX released from the modified, charged hydrogels remained biologically active and had the capability to kill cancer cells. In contrast, control groups of unmodified OPF hydrogels with or without DOX did not exhibit any cytotoxicity. This study demonstrates the feasibility of using SMA-modified OPF hydrogels as a potential carrier for chemotherapeutic drugs for cancer treatments.
The objective of our study was to determine the effects of composite formulation on the compressive modulus and ultimate strength of a biodegradable, in situ polymerizable poly(propylene fumarate) (PPF) and bone fiber scaffold. The following parameters were investigated: the incorporation of bone fibers (either mineralized or demineralized), PPF molecular weight, N-vinyl pyrrolidinone (NVP) crosslinker amount, benzoyl peroxide (BP) initiator amount, and sodium chloride porogen amount. Eight formulations were chosen based on a resolution III two level fractional factorial design. The compressive modulus and ultimate strength of these formulations were measured on a materials testing machine. Absolute values for compressive modulus varied from 21.3 to 271 MPa and 2.8 to 358 MPa for dry and wet samples, respectively. The ultimate strength of the crosslinked composites varied from 2.1 to 20.3 MPa for dry samples and from 0.4 to 16.6 MPa for wet samples. Main effects of each parameter on the measured property were calculated. The incorporation of mineralized bone fibers and an increase in PPF molecular weight resulted in higher compressive modulus and ultimate strength. Both mechanical properties also increased as the amount of benzoyl peroxide increased or the NVP amount decreased in the formulation. Sodium chloride had a dominating effect on the increase of mechanical properties in dry samples but showed little effects in wet samples. Demineralization of bone fibers led to a decrease in the compressive modulus and ultimate strength. Our results suggest that bone fibers are appropriate as structural enforcement components in PPF scaffolds. The desired orthopaedic PPF scaffold might be obtained by changing a variety of composite formulation parameters.
poly(propylene fumarate); bone fiber; orthopaedic biomaterials; injectable; mechanical properties
Comprehensive in vivo biodegradability and biocompatibility of unmodified and Arg-Gly-Asp (RGD) peptide-modified PEG/Sebacic acid based hydrogels were evaluated and compared to the control material poly(lactide-co-glycolide) (PLGA) using a cage implantation system, as well as direct subcutaneous implantation for up to 12 weeks. The total weight loss after 12 weeks of implantation for unmodified PEGSDA and RGD-modified PEGSDA in the cage was approximately 42% and 52%, respectively, with no statistical difference (p> 0.05). The exudate analysis showed that PEGSDA hydrogels induced minimal inflammatory response up to 21 days following implantation, similar to the controls (empty cage and the cage containing PLGA discs). Histology analysis from direct subcutaneous implantation of the hydrogels and PLGA scaffold showed statistically similar resolution of the acute and chronic inflammatory responses with development of the fibrous capsule between the PEGSDA hydrogels and the control (PLGA). The cage system, as well as the histology analysis, demonstrated that the degradation products of both hydrogels, with or without RGD peptide modification, are biocompatible without statistically significant differences in the inflammatory responses, as compared to PLGA.
In vivo biocompatibility; In vivo biodegradation; PEG sebacic acid diacrylate; Hydrogel; RGD-modified hydrogel; Cage implantation
Hydrogels are potentially useful for many purposes in regenerative medicine including drug and growth factor delivery, as single scaffold for bone repair or as a filler of pores of another biomaterial in which host mesenchymal progenitor cells can migrate in and differentiate into matrix-producing osteoblasts. Collagen type I is of special interest as it is a very important and abundant natural matrix component. The purpose of this study was to investigate whether rat bone marrow stromal cells (rBMSCs) are able to adhere to, to survive, to proliferate and to migrate in collagen type I hydrogels and whether they can adopt an osteoblastic fate. rBMSCs were obtained from rat femora and plated on collagen type I hydrogels. Prior to harvest by day 7, 14, and 21, hydrogels were fluorescently labeled, cryo-cut and analyzed by fluorescent-based and laser scanning confocal microscopy to determine cell proliferation, migration, and viability. Osteogenic differentiation was determined by alkaline phosphatase activity. Collagen type I hydrogels allowed the attachment of rBMSCs to the hydrogel, their proliferation, and migration towards the inner part of the gel. rBMSCs started to differentiate into osteoblasts as determined by an increase in alkaline phosphatase activity after two weeks in culture. This study therefore suggests that collagen type I hydrogels could be useful for musculoskeletal regenerative therapies.
Collagen type I hydrogel; bone marrow stromal cells; cell migration; osteogenic differentiation; bone regeneration
We have previously shown experimental transplantation of living allogeneic bone to be feasible without long-term immunosuppression by development of a recipient-derived neoangiogenic circulation within bone. In this study we study the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from DA to PVG rats. Poly(D,L-lactide-co-glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both were inserted intramedullarly along with a recipient-derived a/v bundle. FK-506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic and histomorphometric analysis were performed. Capillary density was greater in the FGF+VEGF group (35.1%) than control (13.9%) (p<0.05), and a linear trend was found from control, FGF, VEGF, to FGF+VEGF (p<0.005). Bone formation rates were greater with VEGF (p<0.01) and FGF+VEGF (p<0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression.
bone; allotransplantation; microspheres; FGF; VEGF
Electrically conductive polymer composites composed of polycaprolactone fumarate and polypyrrole (PCLF-PPy) have been developed for nerve regeneration applications. Here we report the synthesis and characterization of PCLF-PPy and in vitro studies showing PCLF-PPy materials support both PC12 cell and dorsal root ganglia (DRG) neurite extension. PCLF-PPy composite materials were synthesized by polymerizing pyrrole in pre-formed PCLF scaffolds (Mn 7,000 or 18,000 g mol−1) resulting in interpenetrating networks of PCLF-PPy. Chemical compositions and thermal properties were characterized by ATR-FTIR, XPS, DSC, and TGA. PCLF-PPy materials were synthesized with five different anions (naphthalene-2-sulfonic acid sodium salt (NSA), dodecylbenzenesulfonic acid sodium salt (DBSA), dioctyl sulfosuccinate sodium salt (DOSS), potassium iodide (I), and lysine) to investigate effects on electrical conductivity and to optimize chemical composition for cellular compatibility. PCLF-PPy materials have variable electrical conductivity up to 6 mS cm−1 with bulk compositions ranging from 5 to 13.5 percent polypyrrole. AFM and SEM characterization show microstructures with a root mean squared (RMS) roughness of 1195 nm and nanostructures with RMS roughness of 8 nm. In vitro studies using PC12 cells and DRG show PCLF-PPy materials synthesized with NSA or DBSA support cell attachment, proliferation, neurite extension, and are promising materials for future studies involving electrical stimulation.
Electrically Conductive; Polypyrrole; Nerve; PCLF
We present enhanced cell ingrowth and proliferation through crosslinked three-dimensional (3D) nanocomposite scaffolds fabricated using poly(propylene fumarate) (PPF) and hydroxyapatite (HA) nanoparticles. Scaffolds with controlled internal pore structures were produced from computer-aided design (CAD) models and solid freeform fabrication (SFF) technique, while those with random pore structures were fabricated by NaCl leaching technique for comparison. The morphology and mechanical properties of scaffolds were characterized using scanning electron microscopy (SEM) and mechanical testing, respectively. Pore interconnectivity of scaffolds was assessed using X-ray micro-computed tomography (micro-CT) and 3D imaging analysis. In vitro cell studies have been performed using MC3T3-E1 mouse preosteoblasts and cultured scaffolds in a rotating-wall-vessel bioreactor for 4 and 7 days to assess cell attachment, viability, ingrowth depth, and proliferation. The mechanical properties of crosslinked nanocomposite scaffolds were not significantly different after adding HA or varying pore structures. However, pore interconnectivity of PPF/HA nanocomposite scaffolds with controlled pore structures has been significantly increased, resulting in enhanced cell ingrowth depth 7 days after cell seeding. Cell attachment and proliferation are also higher in PPF/HA nanocomposite scaffolds. These results suggest that crosslinked PPF/HA nanocomposite scaffolds with controlled pore structures may lead to promising bone tissue engineering scaffolds with excellent cell proliferation and ingrowth.
Poly(propylene fumarate) (PPF); Hydroxyapatite (HA); Nanocomposite; Solid freeform fabrication (SFF); Pre-osteoblast responses