Magnetic-Responsive Carbon Nanotubes Composite Scaffolds for Chondrogenic Tissue Engineering.

The demand for engineered scaffolds capable of delivering multiple cues to cells continues to grow as the interplay between cell fate with microenvironmental and external cues is revealed. Emphasis has been given to develop stimuli-responsive scaffolds. These scaffolds are designed to sense an external stimulus triggering a specific response (e.g., change in the microenvironment, release therapeutics, etc.) and then initiate/modulate a desired biofunction. Here, magnetic-responsive carboxylated multi-walled carbon nanotubes (cMWCNTs) are integrated into 3D collagen/polylactic acid (PLA) scaffold via a reproducible filtration-based method. The integrity and biomechanical performance of the collagen/PLA scaffolds are preserved after cMWCNT integration. In vitro safety assessment of cMWCNT/collagen/PLA scaffolds shows neither cytotoxicity effects nor macrophage pro-inflammatory response, supporting further in vitro studies. The cMWCNT/collagen/PLA scaffolds enhance chondrocytes metabolic activity while maintaining high cell viability and extracellular matrix (i.e., type II collagen and aggrecan) production. Comprehensive in vitro study applying static and pulsed magnetic field on seeded scaffolds shows no specific cell response in dependence with the applied field. This result is independent of the presence or absence of cMWCNT into the collagen/PLA scaffolds. Taken together, these findings provide additional evidence of the benefits to exploit the CNTs outstanding properties in the design of stimuli-responsive scaffolds.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment.

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.

Gas-foamed poly(lactide-co-glycolide) and poly(lactide-co-glycolide) with bioactive glass fibres demonstrate insufficient bone repair in lapine osteochondral defects.

Deep osteochondral defects may leave voids in the subchondral bone, increasing the risk of joint structure collapse. To ensure a stable foundation for the cartilage repair, bone grafts can be used for filling these defects. Poly(lactide-co-glycolide) (PLGA) is a biodegradable material that improves bone healing and supports bone matrix deposition. We compared the reparative capacity of two investigative macroporous PLGA-based biomaterials with two commercially available bone graft substitutes in the bony part of an intra-articular bone defect created in the lapine femur. New Zealand white rabbits (n = 40) were randomized into five groups. The defects, 4 mm in diameter and 8 mm deep, were filled with neat PLGA; a composite material combining PLGA and bioactive glass fibres (PLGA-BGf); commercial beta-tricalcium phosphate (ß-TCP) granules; or commercial bioactive glass (BG) granules. The fifth group was left untreated for spontaneous repair. After three months, the repair tissue was evaluated with X-ray microtomography and histology. Relative values comparing the operated knee with its contralateral control were calculated. The relative bone volume fraction (∆BV/TV) was largest in the ß-TCP group (p ≤ 0.012), which also showed the most abundant osteoid. BG resulted in improved bone formation, whereas defects in the PLGA-BGf group were filled with fibrous tissue. Repair with PLGA did not differ from spontaneous repair. The PLGA, PLGA-BGf, and spontaneous groups showed thicker and sparser trabeculae than the commercial controls. We conclude that bone repair with ß-TCP and BG granules was satisfactory, whereas the investigational PLGA-based materials were only as good as or worse than spontaneous repair.

The Importance of Controlled Mismatch of Biomechanical Compliances of Implantable Scaffolds and Native Tissue for Articular Cartilage Regeneration.

Scaffolds for articular cartilage repair have to be optimally biodegradable with simultaneous promotion of hyaline cartilage formation under rather complex biomechanical and physiological conditions. It has been generally accepted that scaffold structure and composition would be the best when it mimics the structure of native cartilage. However, a reparative construct mimicking the mature native tissue in a healing tissue site presents a biological mismatch of reparative stimuli. In this work, we studied a new recombinant human type III collagen-polylactide (rhCol-PLA) scaffolds. The rhCol-PLA scaffolds were assessed for their relative performance in simulated synovial fluids of 1 and 4 mg/mL sodium hyaluronate with application of model-free analysis with Biomaterials Enhanced Simulation Test (BEST). Pure PLA scaffold was used as a control. The BEST results were compared to the results of a prior in vivo study with rhCol-PLA. Collectively the data indicated that a successful articular cartilage repair require lower stiffness of the scaffold compared to surrounding cartilage yet matching the strain compliance both in static and dynamic conditions. This ensures an optimal combination of load transfer and effective oscillatory nutrients supply to the cells. The results encourage further development of intelligent scaffold structures for optimal articular cartilage repair rather than simply trying to imitate the respective original tissue.

Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016.

Improved dimensional stability with bioactive glass fibre skeleton in poly(lactide-co-glycolide) porous scaffolds for tissue engineering.

Bone tissue engineering requires highly porous three-dimensional (3D) scaffolds with preferable osteoconductive properties, controlled degradation, and good dimensional stability. In this study, highly porous 3D poly(d,l-lactide-co-glycolide) (PLGA) - bioactive glass (BG) composites (PLGA/BG) were manufactured by combining highly porous 3D fibrous BG mesh skeleton with porous PLGA in a freeze-drying process. The 3D structure of the scaffolds was investigated as well as in vitro hydrolytic degradation for 10weeks. The effect of BG on the dimensional stability, scaffold composition, pore structure, and degradation behaviour of the scaffolds was evaluated. The composites showed superior pore structure as the BG fibres inhibited shrinkage of the scaffolds. The BG was also shown to buffer the acidic degradation products of PLGA. These results demonstrate the potential of these PLGA/BG composites for bone tissue engineering, but the ability of this kind of PLGA/BG composites to promote bone regeneration will be studied in forthcoming in vivo studies.

X-ray microtomography of collagen and polylactide scaffolds in liquids.

Methods to image and assess the microstructure of polymer based biomaterials in liquid phase, for example cell culture medium, are well warranted. X-ray microtomography could provide a mean to visualize and analyze such structures. However, the density of such polymers is close to that of water and hence the X-ray contrast is poor. Moreover, if the biomaterials contain cells and are dried, the cell morphology may be distorted. In this paper we test phosphotungstic acid (PTA) staining to improve the contrast. We imaged collagen and PLA samples with µCT in air, water and alcohol. The methods were compared visually and with contrast to noise ratio calculated from the images. Our results demonstrate that with alcohol the PLA can be imaged also in liquid phase. PTA staining seems to be a good method to increase the contrast for collagen in µCT imaging.

Optical projection tomography can be used to investigate spatial distribution of chondrocytes in three-dimensional biomaterial scaffolds for cartilage tissue engineering.

Biomaterial scaffolds have been used in autologous chondrocyte implantation to facilitate the repair of large lesions and to advance the formation of articular cartilage [Exp. Biol. Med. (Maywood) 237(1) (2012), 10-17]. Biomaterial scaffolds are usually three-dimensional (3-D) porous structures consisting of biodegradable materials to support articular cartilage formation. Adequate porosity of the scaffold is necessary for uniform cell distribution and cell attachment, and the density of the cells in the scaffold should be appropriate for cartilage formation [Cartilage 3(2) (2012), 108-117]. There have been only a restricted number of studies on the spatial distribution of cells in scaffolds, and on the role of this to cartilage formation [J. Biotechnol. 129 (2007), 516-531; Biotechnol. Progr. 14 (1998), 193-202; Biotechnol. Bioeng. 84 (2003), 205-214]. This may be due to the limited availability of appropriate visualization methods. Acquiring 3-D images throughout the scaffold by histology or confocal methods are not applicable to all types of scaffolds, and moreover, they are time consuming, laborious and thus not very feasible for a large scale analysis. To make the visualization of the spatial distribution of the cells easier in biomaterial scaffolds we have applied optical projection tomography (OPT). OPT microscope produces high-resolution 3-D images of both fluorescent and non-fluorescent specimens [Science 296(5567) (2002), 541-545]. Here we demonstrate that the OPT method can be used for the evaluation and visualization of the cell seeding method, spatial distribution and density of cells in biomaterial scaffolds and thus establish the OPT as a valid tool for analysis of cell distribution in cartilage tissue engineering samples.

Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering.

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

Chemical and topographical patterning of hydrogels for neural cell guidance in vitro.

This review focuses on hydrogels and their patterning techniques in relation to central nervous system applications, with emphasis on synthetic and natural materials and chemical and topographical patterning techniques. We describe the properties of hydrogel materials and various techniques used in hydrogel patterning methods. Also, the applicability and utilization of patterned hydrogels with neural cells is discussed. Surface chemistry and topography significantly affect cell behaviour, including cell attachment, migration and maturation. Although several patterning techniques are described in the literature, a review of techniques applicable to hydrogel materials is needed. Use of these patterned cell-hydrogel constructs might provide novel ways to treat central nervous system deficits in the future.

Characterizing and optimizing poly-L-lactide-co-ε-caprolactone membranes for urothelial tissue engineering.

Different synthetic biomaterials such as polylactide (PLA), polycaprolactone and poly-l-lactide-co-ε-caprolactone (PLCL) have been studied for urothelial tissue engineering, with favourable results. The aim of this research was to further optimize the growth surface for human urothelial cells (hUCs) by comparing different PLCL-based membranes: smooth (s) and textured (t) PLCL and knitted PLA mesh with compression-moulded PLCL (cPLCL). The effects of topographical texturing on urothelial cell response and mechanical properties under hydrolysis were studied. The main finding was that both sPLCL and tPLCL supported hUC growth significantly better than cPLCL. Interestingly, tPLCL gave no significant advantage to hUC attachment or proliferation compared with sPLCL. However, during the 14 day assessment period, the majority of cells were viable and maintained phenotype on all the membranes studied. The material characterization exhibited potential mechanical characteristics of sPLCL and tPLCL for urothelial applications. Furthermore, the highest elongation of tPLCL supports the use of this kind of texturing. In conclusion, in light of our cell culture results and mechanical characterization, both sPLCL and tPLCL should be further studied for urothelial tissue engineering.

Comparison of a poly-L-lactide-co-ε-caprolactone and human amniotic membrane for urothelium tissue engineering applications.

The reconstructive surgery of urothelial defects, such as severe hypospadias is susceptible to complications. The major problem is the lack of suitable grafting materials. Therefore, finding alternative treatments such as reconstruction of urethra using tissue engineering is essential. The aim of this study was to compare the effects of naturally derived acellular human amniotic membrane (hAM) to synthetic poly-L-lactide-co-ε-caprolactone (PLCL) on human urothelial cell (hUC) viability, proliferation and urothelial differentiation level. The viability of cells was evaluated using live/dead staining and the proliferation was studied using WST-1 measurement. Cytokeratin (CK)7/8 and CK19 were used to confirm that the hUCs maintained their phenotype on different biomaterials. On the PLCL, the cell number significantly increased during the culturing period, in contrast to the hAM, where hUC proliferation was the weakest at 7 and 14 days. In addition, the majority of cells were viable and maintained their phenotype when cultured on PLCL and cell culture plastic, whereas on the hAM, the viability of hUCs decreased with time and the cells did not maintain their phenotype. The PLCL membranes supported the hUC proliferation significantly more than the hAM. These results revealed the significant potential of PLCL membranes in urothelial tissue engineering applications.

Porous polylactide/beta-tricalcium phosphate composite scaffolds for tissue engineering applications.

Porous polylactide/beta-tricalcium phosphate (PLA/beta-TCP) composite scaffolds were fabricated by freeze-drying. The aim of this study was to characterize these graded porous composite scaffolds in two different PLA concentrations (2 and 3 wt%). Also, three different beta-TCP ratios (5, 10 and 20 wt%) were used to study the effect of beta-TCP on the properties of the polymer. The characterization was carried out by determining the pH, weight change, component ratios, thermal stability, inherent viscosity and microstructure of the scaffolds in 26 weeks of hydrolysis. This study indicated that no considerable change was noticed in the structure of the scaffolds when the beta-TCP filler was added. Also, the amount of beta-TCP did not affect the pore size or the pore distribution in the scaffolds. We observed that the fabrication method improved the thermal stability of the samples. Our results suggest that, from the structural point of view, these scaffolds could have potential for the treatment of osteochondral defects in tissue engineering applications. The porous bottom surface of the scaffold and the increased osteogenic differentiation potential achieved with beta-TCP particles may encourage the growth of bone cells. In addition, the dense surface skin of the scaffold may inhibit the ingrowth of osteoblasts and bone tissue, while simultaneously encouraging the ingrowth of chondrocytes.

Growth and osteogenic differentiation of adipose stem cells on PLA/bioactive glass and PLA/beta-TCP scaffolds.

The aim of this study was to compare the effects of novel three-dimensional composite scaffolds consisting of a bioactive phase (bioactive glass or beta-tricalcium phosphate [beta-TCP] 10 and 20 wt%) incorporated within a polylactic acid (PLA) matrix on viability, distribution, proliferation, and osteogenic differentiation of human adipose stem cells (ASCs). The viability and distribution of ASCs on the bioactive composite scaffolds was evaluated using Live/Dead fluorescence staining, environmental scanning electron microscopy, and scanning electron microscopy. There were no differences between the two concentrations of bioactive glass and beta-TCP in PLA scaffolds on proliferation and osteogenic differentiation of ASCs. After 2 weeks of culture, DNA content and alkaline phosphatase (ALP) activity of ASCs cultured on PLA/beta-TCP composite scaffolds were higher relative to other scaffold types. Interestingly, the cell number was significantly lower, but the relative ALP/DNA ratio of ASCs was significantly higher in PLA/bioactive glass scaffolds than in other three scaffold types. These results indicate that the PLA/beta-TCP composite scaffolds significantly enhance ASC proliferation and total ALP activity compared to other scaffold types. This supports the potential future use of PLA/beta-TCP composites as effective scaffolds for tissue engineering and as bone replacement materials.