Implantation of VEGF transfected preadipocytes improves vascularization of fibrin implants on the cylinder chorioallantoic membrane (CAM) model.

The successful substitution or augmentation of soft tissues by implantation of three dimensional cell constructs, consisting of human preadipocytes and fibrin glue as a carrier matrix, requires a rapid and homogeneous vascularization of the whole implant in order to provide a sufficient blood supply of centrally situated cells. Previous investigations have shown that under in vivo conditions primary human preadipocytes induce vascularization of fibrin matrices by secretion of several growth factors, such as VEGF and bFGF. The current study investigates whether vascularization of implants can be improved by transplantation of preadipocytes following transfection with a VEGF-vector. Transfection was performed by electroporation with an pCMX-GFP and pCMX-VEGF165 vector. Transfection efficiency (GFP expression) and VEGF expression were determined in vitro by FACS analysis and VEGF immunoassay, respectively. In vivo investigations to determine the vascularization of the implants were performed on the cylinder chorioallantoic membrane (CAM). Four million VEGF transfected cells were transferred within a fibrin matrix onto the CAM on the 7(th) day of incubation and after 8 days the vascularization of the implant was histologically examined and evaluated by means of a computer-assisted image analysis program. Transfection of preadipocytes with the GFP vector by electroporation yielded transfection efficiencies between 12% and 41% of surviving cells. Results of the VEGF immunoassay demonstrated that VEGF expression was significantly higher following transfection. Investigations on the CAM outlined a significantly higher rate of vascularization in the transfected vs. control population. Our investigations demonstrate that primary human preadipocytes can be successfully transfected by electroporation with a VEGF vector. The enhanced VEGF expression on transfected cells results in an increase of vascularization of the cell constructs on the CAM.

Fracture healing in the elderly patient.

Clinical experience gives rise to the impression that there are differences in fracture healing in different age groups. It is evident that fractures heal more efficiently in children than in adults. However, minimal objective knowledge exists to evaluate this assumption. Temporal, spatial, and cellular quantitative and qualitative interrelationships, as well as signaling molecules and extracellular matrix have not been comprehensively and adequately elucidated for fracture healing in the geriatric skeleton. The biological basis of fracture healing will provide a context for revealing the pathophysiology of delayed or even impaired bone regeneration in the elderly. We will summarize experimental studies on age-related changes at the cellular and molecular level that will add to the pathophysiological understanding of the compromised bone regeneration capacity believed to exist in the elderly patient. We will suggest why this understanding would be useful for therapeutics focused on bone regeneration, in particular fracture healing at an advanced age.

Adipose precursor cells (preadipocytes) induce formation of new vessels in fibrin glue on the newly developed cylinder chorioallantoic membrane model (CAM).

Successful augmentation of soft tissues by transplantation of preadipocytes within a matrix requires the formation of a new capillary network with connection to the host vessel system. Particularly, cells located centrally within the transplanted cell-matrix-construct represent a population with a blood supply questionable for survival. We demonstrated that under in vivo conditions preadipocytes possess the ability to induce and support the vascularization of the implant presumably by expression of several growth factors, such as VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor). Fertilized White-Leghorn eggs were incubated under standardized conditions. Opening was performed at day three of incubation and preadipocytes with and without recombinant growth factors were transferred into a fibrin matrix and subsequently placed on the Chorioallantoic Membrane (CAM), respectively. Eight days later, the implanted constructs were explanted, histologically processed and vascularization evaluated by means of a computer-assisted image analysis program. Matrices containing preadipocytes displayed a significantly higher density of vascularization, whereas in the control group (fibrin without preadipocytes) no vessel ingrowth was observed. Daily application of recombinant growth factors added to the medium did not positively influence vascularization of the implant. Our investigations demonstrate that preadipocytes possess a strong angiogenic potential to induce and support neovascularization of 3D-fibrin matrices under in vivo conditions. Addition of recombinant growth factors did not result in any stimulatory effect. Neither did the application of fibrin alone demonstrate an angiogenic potential with regard to induction of vascularization.

Engineered adipose tissue supplied by functional microvessels.

A volume-persistent culture of adipose tissue under in vivo conditions can be achieved only by early vascularization after cell transplantation. Cotransplantation of autologous preadipocytes with endothelial cells may enable the early formation of a capillary network. Investigations were performed in vivo in a specially adapted chorioallantoic membrane (CAM) model. Fertilized White Leghorn eggs were incubated and opened on day 3 of incubation and human dermal microvascular endothelial cell (HDMVEC) spheroids and preadipocytes were transferred in a fibrin matrix to the CAM. On day 7 after incubation the composites were explanted and immunohistologically investigated. Numerous vessels consisting of HDMVECs could be detected and the lumena of these vessels were perfused by chick erythrocytes. These results show the formation of a capillary network consisting of transplanted HDMVECs. The microcirculation of chick erythrocytes in vessels consisting of human endothelial cells proves the continuity of a newly formed capillary system to the host vessel system. The experiments demonstrate the first patent connection of tissue-engineered microvessels in adipose tissue to a host vessel system without applying exogenous angiogenic growth factors or transient transfection. The cotransplantation of endothelial cell spheroids with angiogenic mesenchymal cells may lead to the engineering of complex three-dimensional implants.

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model.

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.