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.

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.

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)-ß1 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-ß1 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.

Using the Taguchi method to obtain more finesse to the biodegradable fibers.

The Taguchi method together with Minitab software was used to optimize the melt spun PLLA multifilament fiber finesse. The aim was to minimize the number of spinning experiments to find optimal processing conditions and to maximize the quality of the fibers (thickness, strength, and smoothness). The optimization was performed in two parts. At first, the melt spinning process was optimized considering the drawing that followed and at second step the drawing was optimized. Fine (15 µm) fibers with feasible strength properties (730 MPa) for further processing were produced with the aid of Minitab software.

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 γ-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.

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.

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.

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.

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.

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.

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.

Studies of P(L/D)LA 96/4 non-woven scaffolds and fibres; properties, wettability and cell spreading before and after intrusive treatment methods.

Poly(L/D)lactide 96/4 fibres with diameters of 50 and 80 microm were produced. The smaller diameter fibres were carded and needle punched to form a non-woven mat. Fibres and non-woven mats were hydrolysed for a period of 20 weeks. Fibres and pressed non-woven discs were treated with low-temperature oxygen plasma and alkaline KOH hydrolysis and ethanol washing was used as a reference treatment. The non-wovens lost 50% of their tear strength after 8 weeks in vitro while the fibres still retained 65% tensile strength after 20 weeks. Hydrolysation time in KOH, treatment time and power settings of the oxygen plasma were all directly proportional to the mechanical properties of the fibres. Increasing time (and power) resulted in lower tensile properties. Rapid wetting of the scaffolds was achieved by oxygen plasma, KOH hydrolysation and ethanol washing. Cell culturing using fibroblast cell line was carried out for the treated and non-treated non-woven scaffolds. In terms of adhesion and the spreading of the cells into the scaffold, best results after 3-day culturing were obtained for the oxygen plasma treated scaffolds.

Effect of FGF and polylactide scaffolds on calvarial bone healing with growth factor on biodegradable polymer scaffolds.

Repair of bone defects remains a major concern in reconstructive surgery. Synthetic biodegradable polymers have been used as scaffolds for guided bone regeneration. Fibroblast growth factors (FGFs) promote cell growth, differentiation, and tissue maintenance factors. They can stimulate the proliferation of osteogenic cells and chondrocytes, and also promote angiogenesis. Acidic and basic fibroblast growth factors (FGF-1 and FGF-2, respectively) are the best known members of this protein family. To evaluate the healing of experimental bone defects using poly-L/D-lactide (PLDLA) 96/4 scaffolds and FGF-1, 18 adult rats were operated on. A 6-mm diameter critical size defect (CSD) was made in the calvarial bone of each rat. The animals were divided into three treatment groups: 1) Neither scaffold nor FGF was used (control group); 2) scaffold only; and 3) scaffold with FGF-1. Follow-up time was eight weeks. Samples were embedded in methylmethacrylate and 5-microm thick sections from the middle of each specimen were stained with modified Masson-Goldner method. The shape and size of defects were evaluated radiologically. New bone formation was measured histologically and histomorphometrically. Radiologically, in the control group the shape of the defects changed from round to oval and edges were blunt. In the other groups the defects were round with sharp edges. Histomorphometrically, mean surface area of bone trabeculae was 1.05 mm (SD +/- 0.25) in group 1 (no implant), 1.35 mm (SD +/- 0.52) in group 2 (implant) and 0.79 mm (SD +/- 0.34) in group 3 (implant and FGF-1). Histological examinations revealed no or little osteoid in the groups 1 and 2, whereas in the group 3 samples had little or moderate new bone formation. Accordingly, no clear benefit of using knitted PLDLA scaffolds combined with FGF-1 on the healing of calvarial critical size defects in rats could be demonstrated.

Histologic analysis of bioabsorbable scleral buckling implants: an experimental study on rabbits.

PURPOSE: To analyze histologically tissue reactions to bioabsorbable PLA96 in rabbit eyes. METHODS: Scleral buckling operations were carried out in 48 rabbits. Two materials were used: bioabsorbable PLA96 (polylactide 96/4; L/D molar ratio 96/4) and silicone sponge. One eye of each rabbit was operated on and the other eye served as a nonoperated control. After follow-up times of 1, 3, 5, and 12 months, the rabbits were killed and the eyes enucleated for histology. RESULTS: All rabbits recovered well. Histologically, tissue reactions were very localized; implant fragments were not seen within the sclera. The amounts of fibrous tissue and inflammatory cells (mainly macrophages) inside the implant area increased over time. One rabbit from the silicone group was killed 4 months postoperatively owing to refusal to eat. In the PLA96 group, acute or chronic infections occurred in four rabbits. The bioabsorbable implant was macroscopically easily detectable at 12 months postoperatively. CONCLUSIONS: The PLA96 material used for scleral buckling in rabbits showed good biocompatibility. The material did not undergo biodegradation during the follow-up period of 12 months. PLA96 implants were associated with thicker fibrous tissue encapsulation and more inflammatory cells compared with silicone sponge implants.

Persistence of indentation with bioabsorbable poly-L/D-lactide versus silicone sponge scleral buckling implants.

PURPOSE: To measure the amount and duration of indentation depth achieved with biodegradable poly-L/D-lactide 96/4 (PLA96) and silicone sponge implants. METHODS: Thirty rabbits underwent a scleral buckling procedure. A PLA96 buckling implant was used in 15 rabbits and a silicone sponge buckling implant was in 15 rabbits. A circumferential scleral buckling implant was sutured episclerally on the left eye of each rabbit, just temporal to the superior rectus muscle and 7 mm posterior to the limbus. Computed tomography was performed at 1 week, 3 months, and 5 months after surgery. RESULTS: The PLA96 buckling implant (implant diameter, 3-3.5 mm) used in this study created lower indentation than the silicone sponge implant (implant diameter, 4 mm). The indentation created by the PLA96 implant decreased over time compared with that created by the silicone implant. There were no complications related to either kind of implant. CONCLUSION: Both the silicone sponge implant and the PLA96 implant caused indentation that decreased in a comparable manner over the follow-up period (5 months).