A preliminary study of human amniotic membrane as a potential chondrocyte carrier

Purpose: To investigate the feasibility of using processed human amniotic membrane (HAM) to support the attachment and proliferation of chondrocytes in vitro which in turn can be utilised as a cell delivery vehicle in tissue engineering applications. Methods: Fresh HAM obtained from patients undergoing routine elective caesarean sections was harvested, processed and dried using either freeze drying (FD) or air drying (AD) methods prior to sterilisation by gamma irradiation. Isolated, processed and characterised rabbit autologous chondrocytes were seeded on processed HAM and cultured for up to three weeks. Cell attachment and proliferation were examined qualitatively using inverted brightfield microscopy. Results: Processed HAM appeared to allow cell attachment when implanted with chondrocytes. Although cells seeded on AD and FD HAM did not appear to attach as strongly as those seeded on glycerol preserved intact human amniotic membrane, these cells to be proliferated in cell culture conditions. Conclusion: Preliminary results show that processed HAM promotes chondrocyte attachment and proliferation

In vitro comparative study of white and dark polycaprolactone trifumarate in situ cross-linkable scaffolds seeded with rat bone marrow stromal cells

OBJECTIVE: Dark poly(caprolactone) trifumarate is a successful candidate for use as a bone tissue engineering scaffold. Recently, a white polymeric scaffold was developed that shows a shorter synthesis time and is more convenient for tissue-staining work. This is an in vitro comparative study of both the white and dark scaffolds. METHODS: Both white and dark poly(caprolactone) trifumarate macromers were characterized via Fourier transform infrared spectroscopy before being chemically cross-linked and molded into disc-shaped scaffolds. Biodegradability was assessed by percentage weight loss on days 7, 14, 28, 42 and 56 (n = 5) after immersion in 10% serum-supplemented medium or distilled water. Static cell seeding was employed in which isolated and characterized rat bone marrow stromal cells were seeded directly onto the scaffold surface. Seeded scaffolds were subjected to a series of biochemical assays and scanning electron microscopy at specified time intervals for up to 28 days of incubation. RESULTS: The degradation of the white scaffold was significantly lower compared with the dark scaffold but was within the acceptable time range for bone-healing processes. The deoxyribonucleic acid and collagen contents increased up to day 28 with no significant difference between the two scaffolds, but the glycosaminoglycan content was slightly higher in the white scaffold throughout 14 days of incubation. Scanning electron microscopy at days 1 and 14 revealed cellular growth and attachment. CONCLUSIONS: There was no cell growth advantage between the two forms, but the white scaffold had a slower biodegradability rate, suggesting that the newly synthesized poly(caprolactone) trifumarate is more suitable for use as a bone tissue engineering scaffold

Polycaprolactone-based scaffolds facilitates osteogenic differentiation of human adipose-derived stem cells in a co-culture system

Background: Stem cells in combination with scaffolds and bioactive molecules have made significant contributions to the regeneration of damaged bone tissues. A co-culture system can be effective in enhancing the proliferation rate and osteogenic differentiation of the stem cells. Hence, the aim of this study was to investigate the osteogenic differentiation of human adipose derived stem cells when co-cultured with human osteoblasts and seeded on polycaprolactone (PCL):hydroxyapatite (HA) scaffold; (2) Methods: Human adipose-derived stem cells (ASC) and human osteoblasts (HOB) were seeded in three different ratios of 1:2, 1:2 and 2:1 in the PCL-HA scaffolds. The osteogenic differentiation ability was evaluated based on cell morphology, proliferation rate, alkaline phosphatase (ALP) activity, calcium deposition and osteogenic genes expression levels using quantitative RT-PCR; (3) Results: The co-cultured of ASC/HOB in ratio 2:1 seeded on the PCLHA scaffolds showed the most positive osteogenic differentiation as compared to other groups, which resulted in higher ALP activity, calcium deposition and osteogenic genes expression, particularly Runx, ALP and BSP. These genes indicate that the co-cultured ASC/HOB seeded on PCL-HA was at the early stage of osteogenic development; (4) Conclusions: The combination of co-culture system (ASC/HOB) and PCL-HA scaffolds promote osteogenic differentiation and early bone formation

Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05). The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05), indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading

Fabrication and Characterization of Polycaprolactone (PCL)-Silk-Hydroxyapatite (HA) based scaffold

One of the key aspects that need to be addressed and considered in scaffold fabrication is nanofibres structure. Nanofibrous is one of the most suitable form of artificial matrices for cell growth and proliferation. Electrospinning technique is capable to fabricate polymeric nanofibers with high surface area. Silk, a natural polymer has astounding properties such as biocompatible, biodegradable and bioresorbable. As a result, silk which mimic and analogous to native extracellular matrix (ECM) have been used extensively in the fields of tissue engineering and biomaterials. In the present study, Polycaprolactone (PCL) and Hydroxyapatite (HA) were blended with silk to enhance the mechanical property of the composite scaffold. HA is known for its osteoconductivity while PCL could further enhance the mechanical property of silk. Fourier Transform Infrared (FTIR) spectroscopy and Field Emission Scanning Electron Microscopy (FESEM) methods were used to characterize the PCL-Silk-HA based scaffold. The FESEM results show that the diameter of the fibres were further reduced with the presence of Silk and HA when compared to PCL alone. The FTIR spectra for both PCL-Silk and PCL-Silk-HA based scaffolds revealed the existence of the beta sheet secondary protein structure of the silk protein with the emergence of new characteristic peaks of amide I and amide II at 1645 1/cm and 1488 1/cm respectively. The transmission peak for C-O stretching of PCL based scaffold, at 1206.5 1/cm is observed to have drastically reduced in the PCL-Silk as well as PCL-Silk-HA based scaffold. The phosphate groups are observed at 1131 1/cm, 1000 1/cm and additional phosphate group is shown at 600.87 1/cm peak when HA is presence. These results indicate that all three types of materials have formed a composite structures through chemical bonding. Keywords: biocompatible, biodegradable, bioresorbable, electrospinning, osteoconductivit