Standard fibroblast culture medium usually contains fetal bovine serum (FBS). In theory, unknown risks of infection from bovine disease or immune reaction to foreign proteins may occur if standard culture method is used for future human tissue-engineering development. Human serum (HS) theoretically would be another choice in providing a safer approach and autologous clinically reliable cells.
To evaluate the effect of autologous human serum (AHS) versus pooled human serum (PHS) versus foetal bovine serum (FBS) for growth of articular chondrocytes and formation of chondrocytefibrin constructs.
A potential cure for hearing loss would be to regenerate hair cells by stimulating cells of the damaged inner ear sensory epithelia to proliferate and differentiate into hair cells. Here, we investigated the possibility to isolate, culture-expand and characterize the cells from the cochlea membrane of adult mice. Our results showed that the cultured cells isolated from mouse cochlea membrane were heterogenous in nature. Morphologically there were epithelial like cells, hair cell like, nerve cell like and fibroblastic cells observed in the culture. The cultured cells were immunopositive for specific hair cell markers including Myosin 7a, Calretinin and Espin.
Bone marrow derived progenitor cells have been widely studied for its multipotent property and have proofed to be an important resource in regenerative medicine. However, the propagation of murine bone marrow appeared to be a great challenge as compared to other mammalian species. In this study, various isolation techniques and the plasticity of the isolated cells were evaluated. Our result shows that magnetic sorting technique yielded the most viable cells and displayed wider differentiation capacity.
This study aimed to compare the effects of three different media on the in vivo chondrogenesis of sheep bone marrow stem cells (BMSC). Sheep BMSC were cultured in F12:DMEM + 10% FBS, chondrogenic medium containing 5ng/ml TGF,3 + 50ng/ml IGF-1 and UKM-MECC for three weeks. The cultured cells were then harvested for construct formation with fibrin. Constructed tissues were implanted subcutaneously into nude mice for in vivo development. Cell aggregates were formed in both chondrogenic medium and UKM-MECC demonstrated the early chondrogenesis process. After five weeks of in vivo development, both chondrogenic medium and UKM-MECC promoted cartilage matrix synthesis confirmed by Safranin O staining.
The present work was to determine the development and re-epithelization of bilayered corneal construct (BCC) in vitro and in vivo using scanning electron microscopy (SEM). The in vitro BCC was transplanted to the rabbit's eye and after 90 days the BCC was harvested and analyzed. The corneas were processed for morphology studies. The result indicates that the BICC that was transplanted for 90 days showed good development and re-epithelization of epithelial layer similar to the normal cornea.
Chondrocytes were isolated from normal and microtic human auricular cartilage after ear surgery carried out at Universiti Kebangsaan Malaysia Medical Centre. Chondrocytes were cultured and expanded until passage 4. After reached confluence, cultured chondrocytes at each passage (P1, P2, P3 and P4) were harvested and assigned for growth profile analysis. There was no significant difference in cell viability between both normal and microtic samples (p = 0.84). Both samples showed no significant differences for growth profile parameters in terms of growth rate, population doubling time and total number of cell doubling, except in passage 1, where there is significant difference in cell growth rate (p = 0.004). This preliminary data has indicated that chondrocytes from microtic cartilage has the potential to be used in the reconstruction of human pinna in the future.
Nerve stem cells have a unique characteristic in that they form spherical aggregates, also termed neurospheres, in vitro. The study demonstrated the successful derivation of these neurospheres from bone marrow culture. Their plasticity as nerve stem cells was confirmed. The findings further strengthens the pluripotency of cell populations within the bone marrow.
Human adipose-derived stem cells (HADSC) have demonstrated the capacity of differentiating into bone depending on the specific induction stimuli and growth factors. However, investigation on stem cell characteristic after osteogenic differentiation is still lacking. The goal of this study was to investigate the differential expression of sternness and osteogenic genes in non-induced HADSC compared with HADSC after osteogenic induction using quantitative Real Time RT-PCR. Our results showed that OCT-4, REX-1, FZD9, OSC, RUNX, and ALP were up regulated after osteogenic induction. This may indicated that HADSCs after osteogenic induction still possessed some stemness properties.
Chondrocytes were isolated from articular cartilage biopsy and were cultivated in vitro. Approximately 30 million of cultured chondrocytes per ml were incorporated with autologous plasma-derived fibrin to form three-dimensional construct. Full-thickness punch hole defects were created in lateral and medial femoral condyles. The defects were implanted either with the autologous 'chondrocytes-fibrin' construct (ACFC), autologous chondrocytes (ACI) or fibrin blank (AF). Sheep were euthanized after 12 weeks. The gross morphology of all defects treated with ACFC implantation, ACI and AF exhibited median scores which correspond to a nearly normal appearance according to the International Cartilage Repair Society (ICRS) classification. ACFC significantly enhanced cartilage repair compared to ACI and AF in accordance with the modified O'Driscoll histological scoring scale. The relative sulphated glycosaminoglycans content (%) was significantly higher (p < 0.05) in ACFC when compared to control groups; ACI vs. fibrin only vs. untreated (blank). Results showed that ACFC implantation exhibited superior cartilage-like tissue regeneration compared to ACI. If the result is applicable to the human, it possibly will improve the existing treatment approaches for cartilage restoration in orthopaedic surgery.
Normal tracheal mucociliary clearance is the key to maintaining the health and defense of respiratory airway. Therefore the present of cilia and mucous blanket are important for tracheal epithelium to function effectively. In the present study, we prepared a tissue engineered respiratory epithelium construct (TEREC) made of autologous respiratory epithelium cells, fibroblast and fibrin from sheep owns blood which replaced a created tracheal mucosal defect. Scanning electron microscopy (SEM) showed encouraging result where immature cilia were present on the surface of TEREC. This result indicates that engineered respiratory epithelium was able to function as normal tissue.
This study was to determine if autologous bone marrow mesenchymal stem cells (BMSCs) cultured in chondrogenic medium could repair surgically induced osteoarthritis. Sheep BMSCs were cultured in medium containing 5ng/ml TGFbeta3 + 50ng/ml IGF-1 for three weeks. The cultured cells were then suspended at density of 2x10(6) cell/ml and injected intraarticularly into the osteoarthritic knee joint. After six weeks, the distal head of the femur and the proximal tibial plateau were removed and stained with H&E. The results indicated that knee joints treated with autologous BMSCs cultured in chondrogenic medium showed clear evidence of articular cartilage regeneration in comparison with other groups.
The angiogenic potential of native skin (NS), keratinocytes single skin equivalent (SSE-K), fibroblasts single skin equivalent (SSE-F) and bilayered skin equivalent secreting angiogenic growth factors such as transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) in the in vitro systems at 24, 48, 72 hours and 7 days was compared using Enzyme-Linked Immunosorbent Assay (ELISA). Bilayered skin equivalent exhibit highest release of growth factors within 24 hours to 7 days of culture compared to NS, SSE-K and SSE-F. This proved the potential of bilayered skin equivalent in producing and sustaining growth factors release to enhance angiogenesis, fibroblasts proliferation, matrix deposition, migration and growth of keratinocytes.
A major factor limiting survival following extensive thermal injury is insufficient availability of donor sites to provide enough skin for the required grafting procedures. Limitation of autologous grafting promotes the usage of allograft skin substitutes to promote wound healing. Here, we investigated the wound healing potential of allograft single layered tissue engineered skin which comprises of either keratinocytes (SLTES-K) or fibroblast (SLTES-F) with fibrin as the delivery system. Results from gross and microscopic evaluation showed our single layered tissue engineered skin constructed with keratinocytes or fibroblast after gamma radiation with the dosage of 2Gy could serve as allograft for the treatment of skin loss.
The emergence of tissue engineering and stem cell research has created a tremendous response amongst scientist in Malaysia. However, despite the enthusiastic to embark on the research we have to carefully divert the research towards our needs. This is due to our responsibility to address the mounting problem of communicable diseases here and a very limited funding. As commercialization is a key objective the combination of products towards treating or diagnosing communicable and non-communicable diseases in the developing country is another important factor. The discussion here is mainly on the evolution of tissue engineering in Malaysia and taking a model of tissue engineering in otolaryngology.
Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering.
Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded in vitro. Approximately 30 million cells per ml of cultured chondrocytes were incorporated with autologous plasma-derived fibrin to form a three-dimensional construct. Full-thickness punch hole defects were created in the lateral and medial femoral condyles. The defects were implanted with either an autologous 'chondrocyte-fibrin' construct (ACFC), autologous chondrocytes (ACI) or fibrin blanks (AF) as controls. Animals were killed after 12 weeks. The gross appearance of the treated defects was inspected and photographed. The repaired tissues were studied histologically and by scanning electron microscopy analysis. All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O'Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage. Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI.
This study was aimed at regenerating autologous elastic cartilage for future use in pediatric ear reconstruction surgery. Specific attentions were to characterize pediatric auricular chondrocyte growth in a combination culture medium and to assess the possibility of elastic cartilage regeneration using human fibrin.
Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.
The objectives of this study were to determine the optimum concentration of basic fibroblast growth factor (bFGF) in foetal bovine serum (FBS) or human serum (HS) supplemented medium for adult human nasal septum chondrocyte culture and to evaluate the potential of cartilage regeneration.