Surface-Modified Poly(l-lactide-co-glycolide) Scaffolds for the Treatment of Osteochondral Critical Size Defects-In Vivo Studies on Rabbits.

Poly(l-lactide-co-glycolide) (PLGA) porous scaffolds were modified with collagen type I (PLGA/coll) or hydroxyapatite (PLGA/HAp) and implanted in rabbits osteochondral defects to check their biocompatibility and bone tissue regeneration potential. The scaffolds were fabricated using solvent casting/particulate leaching method. Their total porosity was 85% and the pore size was in the range of 250-320 µm. The physico-chemical properties of the scaffolds were evaluated using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffractometry (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), sessile drop, and compression tests. Three types of the scaffolds (unmodified PLGA, PLGA/coll, and PLGA/HAp) were implanted into the defects created in New Zealand rabbit femoral trochlears; empty defect acted as control. Samples were extracted after 1, 4, 12, and 26 weeks from the implantation, evaluated using micro-computed tomography (µCT), and stained by Masson-Goldner and hematoxylin-eosin. The results showed that the proposed method is suitable for fabrication of highly porous PLGA scaffolds. Effective deposition of both coll and HAp was confirmed on all surfaces of the pores through the entire scaffold volume. In the in vivo model, PLGA and PLGA/HAp scaffolds enhanced tissue ingrowth as shown by histological and morphometric analyses. Bone formation was the highest for PLGA/HAp scaffolds as evidenced by µCT. Neo-tissue formation in the defect site was well correlated with degradation kinetics of the scaffold material. Interestingly, around PLGA/coll extensive inflammation and inhibited tissue healing were detected, presumably due to immunological response of the host towards collagen of bovine origin. To summarize, PLGA scaffolds modified with HAp are the most promising materials for bone tissue regeneration.

Sodium alendronate loaded poly(l-lactide- co-glycolide) microparticles immobilized on ceramic scaffolds for local treatment of bone defects.

Bone tissue regeneration in critical-size defects is possible after implantation of a 3D scaffold and can be additionally enhanced once the scaffold is enriched with drugs or other factors supporting bone remodelling and healing. Sodium alendronate (Aln), a widely used anti-osteoporosis drug, exhibits strong inhibitory effect on bone resorption performed by osteoclasts. Thus, we propose a new approach for the treatment of bone defects in craniofacial region combining biocompatible titanium dioxide scaffolds and poly(l-lactide-co-glycolide) microparticles (MPs) loaded with Aln. The MPs were effectively attached to the surface of the scaffolds' pore walls by human recombinant collagen. Drug release from the scaffolds was characterized by initial burst (24 ± 6% of the drug released within first 24 h) followed by a sustained release phase (on average 5 µg of Aln released per day from Day 3 to Day 18). In vitro tests evidenced that Aln at concentrations of 5 and 2.5 µg/ml was not cytotoxic for MG-63 osteoblast-like cells (viability between 81 ± 6% and 98 ± 3% of control), but it prevented RANKL-induced formation of osteoclast-like cells from macrophages derived from peripheral blood mononuclear cells, as shown by reduced fusion capability and decreased tartrate-resistant acid phosphatase 5b activity (56 ± 5% reduction in comparison to control after 8 days of culture). Results show that it is feasible to design the scaffolds providing required doses of Aln inhibiting osteoclastogenesis, reducing osteoclast activity, but not affecting osteoblast functions, which may be beneficial in the treatment of critical-size bone tissue defects.

Synergistic effect of bimodal pore distribution and artificial extracellular matrices in polymeric scaffolds on osteogenic differentiation of human mesenchymal stem cells.

The main objective of this study was to enhance the biological performance of resorbable polymeric scaffolds for bone tissue engineering. Specifically, we focused on both microstructure and surface modification of the scaffolds to augment adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC). Moreover, a new cell seeding method assuring 90% seeding efficiency on the scaffolds was developed. Poly(l­lactide­co­glycolide) (PLGA) scaffolds with monomodal and bimodal pore distribution were produced by solvent casting/phase separation followed by porogen leaching and modified with artificial extracellular matrices (aECM) consisting of collagen type I and high sulphated hyaluronan (sHya). The application of two porogens resulted in bimodal pore distribution within the PLGA scaffolds as shown by scanning electron microscopy and microcomputer tomography. Two types of pores with diameters 400-600 µm and 2-20 µm were obtained. The scaffolds were successfully coated with a homogenous layer of aECM as shown by Sirius red and toluidine blue staining. In vitro study showed that presence of bimodal pore distribution in combination with collagen/sHya did not significantly influence hMSC proliferation and early osteogenic differentiation compared to scaffolds with monomodal pore distribution. However, it enhanced mineralization as well as the expression of Runt-related transcription factor 2, osteopontin and bone sialoprotein II. As a result PLGA scaffolds with bimodal pore distribution modified with collagen/sHya can be considered as prospective material promoting bone regeneration.

Gentamicin-Loaded Polysaccharide Membranes for Prevention and Treatment of Post-operative Wound Infections in the Skeletal System.

PURPOSE: To develop polysaccharide-based membranes that allow controlled and localized delivery of gentamicin for the treatment of post-operative bone infections. METHODS: Membranes made of gellan gum (GUM), sodium alginate (ALG), GUM and ALG crosslinked with calcium ions (GUM + Ca and ALG + Ca, respectively) as well as reference collagen (COL) were produced by freeze-drying. Mechanical properties, drug release, antimicrobial activity and cytocompatibility of the membranes were assessed. RESULTS: The most appropriate handling and mechanical properties (Young's modulus, E = 92 ± 4 MPa and breaking force, F MAX  = 2.6 ± 0.1 N) had GUM + Ca membrane. In contrast, COL membrane showed F MAX  = 0.14 ± 0.02 N, E = 1.0 ± 0.3 MPa and was deemed to be unsuitable for antibiotic delivery. The pharmacokinetic data demonstrated a uniform and sustainable delivery of gentamicin from GUM + Ca (44.4 ± 1.3% within 3 weeks), while for COL, ALG and ALG + Ca membranes the most of the drug was released within 24 h (55.3 ± 1.9%, 52.5 ± 1.5% and 37.5 ± 1.8%, respectively). Antimicrobial activity against S. aureus and S. epidermidis was confirmed for all the membranes. GUM + Ca and COL membranes supported osteoblasts growth, whereas on ALG and ALG + Ca membranes cell growth was reduced. CONCLUSIONS: GUM + Ca membrane holds promise for effective treatment of bone infections thanks to favorable pharmacokinetics, bactericidal activity, cytocompatibility and good mechanical properties.

Ceramic scaffolds enriched with gentamicin loaded poly(lactide-co-glycolide) microparticles for prevention and treatment of bone tissue infections.

Bone scaffolds are susceptible for bacterial infection when implanted, particularly in compromised bone. Therefore anti-bacterial bone scaffolds are desirable. Here a novel approach to provide bactericidal properties for titanium dioxide scaffolds is proposed. Gentamicin loaded poly(L-lactide-co-glycolide) microparticles were immobilized on the scaffold pore walls by sodium alginate hydrogel. The results show that the microparticles were effectively immobilized on the scaffolds. Desired burst release was observed within the first 8h and gentamicin dose reached 125µg from single scaffold that corresponded to ~25% of total drug introduced in the system. Following the initial burst, the dose was gradually decreasing up to day 10 and afterwards a sustained release of 3µg/day was measured. Cumulatively ~90% of the drug was delivered up to day 50. Above pattern, i.e. burst release with following sustained release, is desired for prevention of perioperative bone infections: burst release stops local infections during post-implantation "decisive period" while further sustained drug release prevents bacterial recolonization. In vitro studies confirmed antimicrobial activity of released gentamicin against Staphylococcus spp. and cytocompatibility of the system with osteoblast-like cells (MG-63). Thus the system is a viable option for the treatment of bone tissue defects.

The influence of sintering conditions on microstructure and mechanical properties of titanium dioxide scaffolds for the treatment of bone tissue defects.

In this study the attempts to improve mechanical properties of highly-porous titanium dioxide scaffolds produced by polymer sponge replication method were investigated. Particularly the effect of two-step sintering at different temperatures on microstructure and mechanical properties (compression test) of the scaffolds were analysed. To this end microcomputed tomography and scanning electron microscopy were used as analytical methods. Our experiments showed that the most appropriate conditions of manufacturing were when the scaffolds were heat-treated at 1500 °C for 1 h followed by sintering at 1200 °C for 20 h. Such scaffolds exhibited the highest compressive strength which was correlated with the highest linear density and the lowest size of grains. Moreover, grain size distribution was narrower with predominating fraction of fine grains 10-20 µm in size. Smaller grains and higher linear density sug- gested that in this case densification process prevailed over undesirable process of grain coarsening, which finally resulted in im- proved mechanical properties of the scaffolds.

Resorbable scaffolds modified with collagen type I or hydroxyapatite: in vitro studies on human mesenchymal stem cells.

Poly(L-lactide-co-glycolide) (PLGA) scaffolds of pore size within the range of 250-320 µm were produced by solvent casting/ porogen leaching method. Afterwards, they were modified through adsorption of collagen type I and incubation in simulated body fluid (SBF) to allow deposition of hydroxyapatite (HAp). The wettability of the scaffolds was measured by sessile drop test. Scanning electron microscopy (SEM) evaluation and energy dispersive X-ray analysis (EDX) were also performed. SEM evaluation and EDX analysis depicted the presence of HAp deposits and a collagen layer on the pore walls on the surface and in the bulk of the scaffolds. Wettability and water droplets penetration time within the scaffolds decreased considerably after applying modifications. Human mesenchymal stem cells (hMSC) were cultured on the scaffolds for 28 days and cell morphology, proliferation and differentiation as well as calcium deposition were evaluated. Lactate dehydrogenase (LDH) activity results revealed that cells cultured on tissue culture polystyrene (TCPS) exhibited high proliferation capacity. Cell growth on the scaffolds was slower in comparison to TCPS and did not depend on modification applied. On the other hand, osteogenic differentiation of hMSC as confirmed by alkaline phosphatase (ALP) activity and mineralization results was enhanced on the scaffolds modified with hydroxyapatite and collagen.