Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin Histological and immunohistochemical examination / 中国组织工程研究

BACKGROUND: Tissue engineering is a multidisciplinary area of research which aims to regenerate damaged tissues and organs in the human body, starting from the assumption that almost all animal tissues in the human body can be cultivated in a laboratory. The general principle is to isolate stem cells from a patient who requires a transplant and then the cells are cultured to grow and differentiate on a suitable support to produce the replacement tissue. On the one hand, it is necessary to find a suitable support (matrix or scaffold) on which cells can adhere and form stratified structures. On the other hand, the conditions allowing cells to proliferate and differentiate into the various types of tissues must be understood and reproduced. At present, the materials used as support in the treatment of ulcers and bums are manifold, such as collagen, hyaluronic acid, fibrin and PLGA. OBJECTIVE: To discuss the effectiveness of a new kind of biomaterial, autologous plasma, as scaffold for the growth, expansion and differentiation of autologous fibroblasts and keratinocytes.DESIGN: We have devised a method for dermal regeneration using autologous fibroblasts immersed in a matrix of human plasma so as to eliminate any problem linked to the safety of the sample and rejection from a patient. On this new dermis, keratinocytes have then been seeded.TIME AND SETTING: This work was performed at Cell Factory of C. Poma Hospital in Mantova in 2008. PARTICIPANT/MATERIALS: The human keratinocytes and fibroblasts used for this research were taken from a skin fragment after mastectomy in a 58-year-old patient. The experiment was approved by the independent ethics committee of C. Poma Hospital and the patient was requested to give informed consent.INTERVENTIONS/METHODS: Our studies were focussed on the isolation of human basal keratinocytes and fibroblasts from autologous biopsied skin samples and on their proliferation in culture flasks. Autologous plasma was subsequently used as scaffold for the formation of skin grafts whose morphofunctional and immunohistochemical characteristics are similar to those of normal skin.MAIN OUTCOME MEASURES: In order to evaluate the results, the samples of plasma were fixed in formalin, embedded in paraffin, stained with haematoxilyn-eosin and viewed through a microscope to count the number of cells in 40-fold 5 fields. Furthermore, all specimens underwent immunohistochemical examination for vimentin, keratin AE1 and AE3, collagen IV, P63 and Mib 1. RESULTS: The skin grafts obtained in the present study consist of few layers of normally-shaped keratinocytes on a plasma matrix with fibroblasts embedded inside. The formation of basement membrane shows that autologous plasma is a good scaffold for the growth, differentiation and proliferation of keratinocytes and fibroblasts and it also demonstrates that dermal and epidermal cells can be interlocked to reconstruct artificial grafts identical to normal skin.CONCLUSION: Plasma has given a remarkable performance as scaffold for the proliferation and differentiation of keratinocytes and fibroblasts. In particular, we have observed that fibroblast-enriched plasma can replace dermis temporarily, besides being a robust scaffold for the growth of keratinocytes. In addition, plasma is relatively cheap and easy to prepare. The use of autologous keratinocytes and fibroblasts, as well as that of autologous plasma, is essential to avoid any type of rejection. This kind of therapy is ideal for patients with chronic defects requiring continuous grafts.

Tissue engineering: chondrocyte culture on type 1 collagen support. Cytohistological and immunohistochemical study.

The scope of our study is to evaluate the possibility of cultivating and expanding human chondrocytes and seeding them on pure equine type I collagen support. Our results show that human articular cartilaginous cells can multiply and grow on type I collagen substrate with production of extracellular matrix. This type of chondrocyte culture on a support can be used for repairing cartilaginous lesions since they show a correct morphology (evaluated by cytological and histological methods) and a suitable differentiation and phenotype as shown by Alcian PAS staining to indicate the presence of mucopolysaccharides, and immunohistochemical methods to identify collagen II. We believe that these chondrocyte cultures on this biomaterial can be used for repairing cartilaginous lesions with improvement of surgical technique; the support allows adhesion of the chondrocytes to the cartilaginous lesion and a mallebility that favours optimum spatial adaptation.

FLICE/caspase-8 activation triggers anoikis induced by beta1-integrin blockade in human keratinocytes.

Beta1-integrin protects keratinocyte stem cells (KSC) from cell-detachment apoptosis ('anoikis'). Here we show that caspase-8 active protein is detected in both young transit amplifying (TA) cells and TA cells, but not in KSC. On suspension, caspases are activated earlier in young TA than in KSC, whereas anti-beta1-integrin neutralizing antibody accelerates caspase activation in both KSC and young TA. Caspases 8 and 10 are the first caspases to be activated whereas caspase-8 inhibitor zIETD-fmk delays the activation of Bid, caspase-9 and caspase-3. However, the caspase-9 inhibitor zLEDH-fmk does not block the activation of caspase-8, Bid, caspase-10 and caspase-3. Moreover, caspase-8, but not caspase-9 inhibitor partially prevents keratinocyte anoikis. As FLIP inhibits caspase-8 processing, we retrovirally infected HaCaT keratinocytes with c-FLIP(L). Anti-beta1-integrin fails to activate caspase-8, Bid, caspase-9 and to induce the release of cytochrome c in c-FLIP(L) overexpressing keratinocytes. Finally, overexpression of c-FLIP(L) partially prevents anoikis in both suspended and anti-beta1 integrin-treated cells. Taken together, these results indicate that the extrinsic apoptotic pathway triggered by caspase-8 predominates in keratinocyte anoikis. However, the release of cytochrome c and the later activation of caspase-9 seem to suggest that the intrinsic mitochondrial pathway may intervene as a positive feedback loop of caspase activation.

Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner.

Because inhibition of integrin signaling induces apoptosis, we investigated whether keratinocytes expressing beta1 and alpha6beta4 integrins (enriched for stem cells) are protected from cell death. Keratinocytes rapidly adhering to type IV collagen expressed highest levels of beta1 and alpha6beta4 and of the anti-apoptotic stem cell marker p63. Apoptotic cells were significantly higher in slowly adhering than in rapidly adhering keratinocytes. Anti-beta1 integrin caused a significant increase in apoptotic cells, while it decreased Bcl-2 levels in stem keratinocytes. Bax and Bad proteins were higher in slowly adhering than in rapidly adhering cells. By contrast, Bcl-2, Bcl-x and Mcl-1 proteins were highest in rapidly adhering keratinocytes and nearly absent in slowly adhering cells. After addition of anti-beta1 integrin, the apoptotic rate was significantly higher in HaCaT cells not expressing Bcl-2 than in controls. These results indicate that keratinocytes enriched for stem cells are protected from apoptosis via beta1 integrin, in a Bcl-2 dependent manner.