Ville Ellä

Growth and Osteogenic Differentiation of Adipose Stem Cells on PLA/Bioactive Glass and PLA/β-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.

Fibrin-polylactide-based tissue-engineered vascular graft in the arterial circulation

There is a clear clinical requirement for the design and development of living, functional, small-calibre arterial grafts. Here, we investigate the potential use of a small diameter, tissue-engineered artery in a pre-clinical study in the carotid artery position of sheep. Small-calibre ( approximately 5 mm) vascular composite grafts were molded using a fibrin scaffold supported by a poly(L/D)lactide 96/4 (P(L/D)LA 96/4) mesh, and seeded with autologous arterial-derived cells prior to 28 days of dynamic conditioning. Conditioned grafts were subsequently implanted for up to 6 months as interposed carotid artery grafts in the same animals from which the cells were harvested. Explanted grafts (n = 6) were patent in each of the study groups (1 month, 3 months, 6 months), with a significant stenosis in one explant (3 months). There was a complete absence of thrombus formation on the luminal surface of grafts, with no evidence for aneurysm formation or calcification after 6 months in vivo. Histological analyses revealed remodeling of the fibrin scaffold with mature autologous proteins, and excellent cell distribution within the graft wall. Positive vWf and eNOS staining, in addition to scanning electron microscopy, revealed a confluent monolayer of endothelial cells lining the luminal surface of the grafts. The present study demonstrates the successful production and mid-term application of an autologous, fibrin-based small-calibre vascular graft in the arterial circulation, and highlights the potential for the creation of autologous implantable arterial grafts in a number of settings.

Tissue-Engineered Small-Caliber Vascular Graft Based on a Novel Biodegradable Composite Fibrin-Polylactide Scaffold

Small-caliber vascular grafts (< or =5 mm) constructed from synthetic materials for coronary bypass or peripheral vascular repair below the knee have poor patency rates, while autologous vessels may not be available for harvesting. The present study aimed to create a completely autologous small-caliber vascular graft by utilizing a bioabsorbable, macroporous poly(L/D)lactide 96/4 [P(L/D)LA 96/4] mesh as a support scaffold system combined with an autologous fibrin cell carrier material. A novel molding device was used to integrate a P(L/D)LA 96/4 mesh in the wall of a fibrin-based vascular graft, which was seeded with arterial smooth muscle cells (SMCs)/fibroblasts and subsequently lined with endothelial cells. The mold was connected to a bioreactor circuit for dynamic mechanical conditioning of the graft over a 21-day period. Graft cell phenotype, proliferation, extracellular matrix (ECM) content, and mechanical strength were analyzed. alpha-SMA-positive SMCs and fibroblasts deposited ECM proteins into the graft wall, with a significant increase in both cell number and collagen content over 21 days. A luminal endothelial cell lining was evidenced by vWf staining, while the grafts exhibited supraphysiological burst pressure (>460 mmHg) after dynamic cultivation. The results of our study demonstrated the successful production of an autologous, biodegradable small-caliber vascular graft in vitro, with remodeling capabilities and supraphysiological mechanical properties after 21 days in culture. The approach may be suitable for a variety of clinical applications, including coronary artery and peripheral artery bypass procedures.

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.

Use of adipose stem cells and polylactide discs for tissue engineering of the temporomandibular joint disc

There is currently no suitable replacement for damaged temporomandibular joint (TMJ) discs after discectomy. In the present study, we fabricated bilayer biodegradable polylactide (PLA) discs comprising a non-woven mat of poly(L/D)lactide (P(L/D)LA) 96/4 and a P(L/DL)LA 70/30 membrane plate. The PLA disc was examined in combination with adipose stem cells (ASCs) for tissue engineering of the fibrocartilaginous TMJ disc in vitro. ASCs were cultured in parallel in control and chondrogenic medium for a maximum of six weeks. Relative expression of the genes, aggrecan, type I collagen and type II collagen present in the TMJ disc extracellular matrix increased in the ASC-seeded PLA discs in the chondrogenic medium. The hypertrophic marker, type X collagen, was moderately induced. Alcian blue staining showed accumulation of sulphated glycosaminoglycans. ASC differentiation in the PLA discs was close to that observed in pellet cultures. Comparison of the mRNA levels revealed that the degree of ASC differentiation was lower than that in TMJ disc-derived cells and tissue. The pellet format supported the phenotype of the TMJ disc-derived cells under chondrogenic conditions and also enhanced their hyalinization potential, which is considered part of the TMJ disc degeneration process. Accordingly, the combination of ASCs and PLA discs has potential for the development of a tissue-engineered TMJ disc replacement.

Human coronary artery smooth muscle cell response to a novel PLA textile/fibrin gel composite scaffold.

Previous studies have demonstrated the potential of fibrin as a cell carrier for cardiovascular tissue engineering applications. Unfortunately, fibrin exhibits poor mechanical properties. One method of addressing this issue is to incorporate a textile in fibrin to provide structural support. However, it is first necessary to develop a deeper understanding of the effect of the textile on cell response. In this study, the cytotoxicity of a polylactic acid (PLA) warp-knit textile was assessed with human coronary artery smooth muscle cells (HCASMC). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was employed to examine the gene expression of HCASMC embedded in fibrin with and without the textile. Five genes were examined over a 3-week period: smooth muscle alpha-actin (SMalphaA), myosin heavy chain 11 smooth muscle (SM1/SM2), calponin, myosin heavy chain 10 non-muscle (SMemb) and collagen. Additionally, a microarray analysis was performed to examine a wider range of genes. The knitting process did not adversely affect the cell response; there was no dramatic change in cell number or metabolic rate compared to the negative control. After 3 weeks, there was no significant difference in gene expression, except for a slight decrease of 10% in SMemb in the fibrin with textile. After 3 weeks, there were no obvious cytotoxic effects observed as a result of the knitting process and the gene expression profile did not appear to be altered in the presence of the mesh in the fibrin gel.

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

Porous polylactide/β‐tricalcium phosphate (PLA/β‐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 β‐TCP ratios (5, 10 and 20 wt%) were used to study the effect of β‐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 β‐TCP filler was added. Also, the amount of β‐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 β‐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. Copyright

Autologous adipose stem cells and polylactide discs in the replacement of the rabbit temporomandibular joint disc

The temporomandibular joint (TMJ) disc lacks functional replacement after discectomy. We investigated tissue-engineered bilayer polylactide (PLA) discs and autologous adipose stem cells (ASCs) as a potential replacement for the TMJ disc. These ASC discs were pre-cultured either in control or in differentiation medium, including transforming growth factor (TGF)-β1 for one week. Prior to implantation, expression of fibrocartilaginous genes was measured by qRT-PCR. The control and differentiated ASC discs were implanted, respectively, in the right and left TMJs of rabbits for six (n = 5) and 12 months (n = 5). Thereafter, the excised TMJ areas were examined with cone beam computed tomography (CBCT) and histology. No signs of infection, inflammation or foreign body reactions were detected at histology, whereas chronic arthrosis and considerable condylar hypertrophy were observed in all operated joints at CBCT. The left condyle treated with the differentiated ASC discs appeared consistently smoother and more sclerotic than the right condyle. The ASC disc replacement resulted in dislocation and morphological changes in the rabbit TMJ. The ASC discs pre-treated with TGF-β1 enhanced the condylar integrity. While adverse tissue reactions were not shown, the authors suggest that with improved attachment and design, the PLA disc and biomaterial itself would hold potential for TMJ disc replacement.

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.

Knitted polylactide 96/4 L/D structures and scaffolds for tissue engineering: Shelf life, in vitro and in vivo studies

This study covers the whole production cycle, from biodegradable polymer processing to an in vivo tissue engineered construct. Six different biodegradable polylactide 96/4 L/D single jersey knits were manufactured using either four or eight multifilament fiber batches. The properties of those were studied in vitro for 42 weeks and in 0- to 3-year shelf life studies. Three types (Ø 12, 15 and 19 mm) of cylindrical scaffolds were manufactured from the knit, and the properties of those were studied in vitro for 48 weeks. For the Ø 15 mm scaffold type, mechanical properties were also studied in a one-year in vivo experiment. The scaffolds were implanted in the rat subcutis. All the scaffolds were g-irradiated prior to the studies. In vitro, all the knits lost 99% of their mechanical strength in 30 weeks. In the three-year follow up of shelf life properties, there was no decrease in the mechanical properties due to the storage time and only a 12% decrease in molecular weight. The in vitro and in vivo scaffolds lost their mechanical properties after 1 week. In the case of the in vivo samples, the mechanical properties were restored again, stepwise, by the presence of growing/maturing tissue between weeks 3 and 12. Faster degradation was observed with in vitro scaffolds compared to in vivo scaffolds during the one-year follow up.