Electrospun hydroxyapatite-containing chitosan nanofibers crosslinked with genipin for bone tissue engineering applications

Reconstruction of large bone defects remains problematic in orthopedic and craniofacial clinical practice. Engineering a functional scaffold mimicking the architectural structure of bone and the regenerative capacity of periosteum offers a potential solution. Electrospun nanofibrous scaffolds are superior in surface area, biomimetic properties, and architecture for the proliferation, and differentiation of osteoprogenitor cells. Chitosan (CTS), the deacetylated form of chitin found in the exoskeleton of crustaceans, is a versatile biomaterial with structural similarity to the extracellular matrix (ECM) of bone.In this thesis, the need of fabricating a regenerative material for bone tissue engineering is addressed by demonstrating the ability of genipin crosslinked electrospun chitosan (CTS-GP) nanofibers mineralized with hydroxyapatite (CTS-HA-GP) to act as osteogenic templates capable of supporting osteoblast adhesion and differentiation with the potential to induce mesenchymal stem cell differentiation and craniofacial regeneration in vivo. Fibrous scaffolds with average fiber diameters of 227±154 nm as spun and 335±119 nm after crosslinking with genipin were generated. Physical, chemical and mechanical analyses were performed for scaffold characterization as well as cytocompatability and osteogenic expression of 7F2 mouse osteoblasts to demonstrate the ability of these scaffolds to support functional osteoblasts. Induction was also observed by the capacity of these scaffolds to induce osteogenic differentiation of human bone-marrow derived mesenchymal stem cells in vitro. Finally, the osseointegrative capacity of these scaffolds was observed by in vivo implantation into a murine calvarial defect model, demonstrating no immunorejection and the early presence of calcified tissue formation.Furthermore the need for enhanced porosity in electrospun scaffolds to induce proper cell infiltration into the scaffold using mesenchymal stem cells on PLGA scaffolds with sacrificed gelatin fibers was also explored. PLGA was used to look into optimizing an appropriate scaffold for bone regeneration due to its ease in fabrication and manipulation by observing mechanical properties and porosity at different ratios compared with cell infiltration. However, it was observed that the gelatin was not being removed as hypothesized and was rather modifying the physical properties of the scaffold. A mathematical model was developed to describe cell proliferation across the scaffolds and cell infiltration into the scaffold.Ph.D., Biomedical Science -- Drexel University, 201

Dose Responsive Effects of Subcutaneous Pentosan Polysulfate Injection in Mucopolysaccharidosis Type VI Rats and Comparison to Oral Treatment

<div><p>Background</p><p>We previously demonstrated the benefits of daily, oral pentosan polysulfate (PPS) treatment in a rat model of mucopolysaccharidosis (MPS) type VI. Herein we compare these effects to once weekly, subcutaneous (sc) injection. The bioavailability of injected PPS is greater than oral, suggesting better delivery to difficult tissues such as bone and cartilage. Injected PPS also effectively treats osteoarthritis in animals, and has shown success in osteoarthritis patients.</p><p>Methodology/Principal Findings</p><p>One-month-old MPS VI rats were given once weekly sc injections of PPS (1, 2 and 4 mg/kg, human equivalent dose (HED)), or daily oral PPS (4 mg/kg HED) for 6 months. Serum inflammatory markers and total glycosaminoglycans (GAGs) were measured, as were several histological, morphological and functional endpoints. Overall, weekly sc PPS injections led to similar or greater therapeutic effects as daily oral administration. Common findings between the two treatment approaches included reduced serum inflammatory markers, improved dentition and skull lengths, reduced tracheal deformities, and improved mobility. Enhanced effects of sc treatment included GAG reduction in urine and tissues, greater endurance on a rotarod, and better improvements in articular cartilage and bone in some dose groups. Optimal therapeutic effects were observed at 2 mg/kg, sc. No drug-related increases in liver enzymes, coagulation factor abnormalities or other adverse effects were identified following 6 months of sc PPS administration.</p><p>Conclusions</p><p>Once weekly sc administration of PPS in MPS VI rats led to equal or better therapeutic effects than daily oral administration, including a surprising reduction in urine and tissue GAGs. No adverse effects from sc PPS administration were observed over the 6-month study period.</p></div

Pentosan Polysulfate: Oral Versus Subcutaneous Injection in Mucopolysaccharidosis Type I Dogs.

BackgroundWe previously demonstrated the therapeutic benefits of pentosan polysulfate (PPS) in a rat model of mucopolysaccharidosis (MPS) type VI. Reduction of inflammation, reduction of glycosaminoglycan (GAG) storage, and improvement in the skeletal phenotype were shown. Herein, we evaluate the long-term safety and therapeutic effects of PPS in a large animal model of a different MPS type, MPS I dogs. We focused on the arterial phenotype since this is one of the most consistent and clinically significant features of the model.Methodology/principal findingsMPS I dogs were treated with daily oral or biweekly subcutaneous (subQ) PPS at a human equivalent dose of 1.6 mg/kg for 17 and 12 months, respectively. Safety parameters were assessed at 6 months and at the end of the study. Following treatment, cytokine and GAG levels were determined in fluids and tissues. Assessments of the aorta and carotid arteries also were performed. No drug-related increases in liver enzymes, coagulation factors, or other adverse effects were observed. Significantly reduced IL-8 and TNF-alpha were found in urine and cerebrospinal fluid (CSF). GAG reduction was observed in urine and tissues. Increases in the luminal openings and reduction of the intimal media thickening occurred in the carotids and aortas of PPS-treated animals, along with a reduction of storage vacuoles. These results were correlated with a reduction of GAG storage, reduction of clusterin 1 staining, and improved elastin integrity. No significant changes in the spines of the treated animals were observed.ConclusionsPPS treatment led to reductions of pro-inflammatory cytokines and GAG storage in urine and tissues of MPS I dogs, which were most evident after subQ administration. SubQ administration also led to significant cytokine reductions in the CSF. Both treatment groups exhibited markedly reduced carotid and aortic inflammation, increased vessel integrity, and improved histopathology. We conclude that PPS may be a safe and useful therapy for MPS I, either as an adjunct or as a stand-alone treatment that reduces inflammation and GAG storage

Flexural mechanics of normal, untreated MPS VI, and PPS-treated MPS VI femurs.

<p>The whole bone stiffness (<b>A</b>) and flexural modulus (<b>B</b>) were decreased in MPS VI compared to normal animals, and no changes were observed in the PPS treatment groups. However, force at break (<b>C</b>) and flexural strength (<b>D</b>) did reveal increasing improvements in a dose dependent manner, indicating that sc PPS treatment moderately enhanced the fracture strength of the femurs.</p

GAG reduction in PPS-treated MPS VI rats.

<p>(<b>A</b>) Total urine GAGs were significantly reduced in all PPS-treated MPS VI rat groups compared to untreated MPS control animals, regardless of the mode of administration. Subcutaneous treatment also significantly reduced urine GAGs compared to oral treatment. Tissue GAGs were significantly reduced only in the MPS VI rats treated with sc PPS. (<b>B</b>) kidney, (<b>C</b>) liver and (<b>D</b>) spleen. A dose responsive reduction was observed in the kidney and liver. *P<0.05 comparing treated to untreated MPS VI rats. ** P<0.05 comparing sc to oral treatment.</p

Histological analysis of MPS VI rat femoral growth plates and articular cartilage.

<p>Representative images are shown for untreated MPS VI rats and each of the sc treatment groups (H & E staining, 20× magnification). (<b>A</b>) Growth plate analyses. Seven-month-old MPS VI rats revealed a complete loss of columnar arrangement in the knee growth plates as compared to normal animals. Overall, PPS sc treatment resulted in moderately improved chondrocyte orientation and growth plate organization, although significant vacuolated chondrocytes were still observed. <b>*</b>Indicates the growth plate. (<b>B</b>) Articular cartilage analyses. Micrographs revealed that sc PPS treatment reduced vacuole formation, suggesting reduced GAG storage in MPS VI articular chondrocytes, in a dose dependent manner. Weekly sc injections of 1 mg/kg had little to no effect, while improvements can be seen at 2 and 4 mg/kg.</p

Motor activity in PPS treated MPS VI rats.

<p>Two weeks after the last PPS dose, treated MPS VI animals were subjected to rotarod analysis at five different speeds (range: 20–40 revolutions per min (RPM)), and their performance was compared to normal and untreated, age and gender-matched animals. Black columns represent untreated MPS VI rats; white hatched columns, oral 4 mg/kg PPS-treated MPS VI rats; light gray columns, 4 mg/kg sc PPS-treated MPS VI rats; dark gray columns, 2 mg/kg sc PPS-treated MPS VI rats; grey hatched columns, and 1 mg/kg sc PPS-treated MPS VI rats. All groups of treated MPS VI rats (oral and sc) remained on the rotating rod significantly longer than untreated animals (p<0.005). At the higher speeds (30, 35 and 40 RPM) MPS VI animals receiving all sc PPS doses had significantly better endurance and remained on the apparatus longer than those receiving oral PPS (p<0.05).</p

Spinal trabecular analysis of PPS-treated MPS VI rats.

<p>3-D microCT reconstructions of (<b>A</b>) normal, (<b>B</b>) untreated MPS VI, (<b>C</b>) 1 mg/kg, (<b>D</b>) 2 mg/kg, and (<b>E</b>) 4 mg/kg sc treated T1 thoracic vertebrae. All images were taken at the point where the pedicle connected with the spinal body. As seen in the untreated MPS VI image (<b>B</b>), a significant amount of dense cortical tissue (indicated by a whiter color intensity) is present compared to the normal animal (<b>A</b>). The cortical tissue also appeared to grow into the trabecular area in MPS VI animals (indicated by arrows and arrowheads). The 2 and 4 mg/kg PPS doses reduced the cortical in-growth and promoted the formation of better-oriented trabeculae in the MPS VI rat vertebrae. P values to represent statistical differences are presented as * for p<0.05 and ** for p<0.01.</p