Pre-shaped Large-volume Engineered Vascularized Pedicled Adipose Flaps In A Rabbit Model:a Two Stage Tissue Engineering Chamber-based Procedure

Background and Objection:Large soft-tissue defects caused by congenital deformity, trauma, or tumor resection require complex reconstruction, which is a major challenge for the plastic surgeons. Autologous vascularized tissue transfer is the most widely accepted and recommended therapy. However, this surgical procedure involves many technical challenges and inevitably leads to donor-site morbidity.Adipose tissue engineering is a growing field that aims to provide a unique alternative to autologous tissue transplantation for the reconstruction of soft-tissue defects. In 2007, researchers found that when an adipose flap based on the superficial inferior epigastric vascular pedicle was placed into a hollow chamber that was implanted subcutaneously in the groin of a rat, significant adipose growth resulted. This tissue-engineering chamber (TEC) technique introduced the new possibilities in tissue engineering.Previous studies showed that large volume adipose tissues could be produced in TECs. However, long-term observations with large animal models reveal that the implanted adipose flaps do not grow sufficiently to take the 100% space of the TEC, which means that the volume of engineered adipose flaps is not maximized. Our studies with rats supply a possible reason for this. We found that when a TEC is placed in animals, the newly formed tissue is covered by a thick capsule. This capsule may reduce the perfusion of the surrounded tissue and restrict further growth. Therefore, we speculate that the capsule may be a key mechanism that prevents engineered tissue flaps from reaching their maximal volume. However, the capsule may also have a positive effect:even though it may restrict the flap volume, it also provides the flap with its shape, which is determined by the contours of the TEC.According to previous reports, we know that the adipose flaps Morrison’s team constructed are mostly round. However, defects in clinical are various shapes. We have the following questions. First of all, whether different shapes adipose flaps can be constructed in different shapes TECs? Then how to construct larger volume flaps to meet clinical needs? And the last one is the long-term stability of de novo engineered adipose flaps.On the basis of these observations, we hypothesized that large TEC-shaped adipose tissue flaps could be generated by employing a two-stage TEC-based procedure. The first step is to grow the pedicled adipose tissue flap in a TEC with the appropriate shape until the flap volume stabilizes. The second step is to remove the TEC, thereby promoting the regrowth of the pre-shaped adipose flap. To test this two-step technique, pedicled adipose tissue flaps were grown in differently shaped TECs in a rabbit model and the gross and histological changes to the flaps that occurred during this process were observed.Methods1. Prefabricate specific shape large volume engineered pedicled adipose tissues flapTwo TECs with round and heart shapes were implanted subcutaneously in the dorsal area of rabbits bilaterally, after which adipose flaps with axis vessel pedicles were inserted into the TECs. Rabbits were sacrificed at 2,4 and 8 weeks for constructs volume measurement, shape assessment, and histological analysis.2. Two stage Tissue Engineering Chamber-based growth patternChambers were removed when constructs in TECs stabilized. After 4 weeks, we measured the constructs volume again and observed histologic changes to explore the feasibility of two stage growth pattern.3. Long-term stability of the TEC-based constructsAfter mature and stable consructs induced in TECs, one of the two flaps in each rabbit dorsa underwent chamber removal. The other flap remained in chamber. Changes in the two groups were observed at 16 weeks and 24 weeks.4. Statistical analysisResults were expressed as the mean standard error. SPSS 13.0 software was used for data analysis. If statistical significance was reached, an independent Bonferroni’s test of two groups at one time point and ANOVA with post-hoc analysis of Student’s t-test of one group at all time points were performed. A value of p< 0.05 was considered to indicate statistical significance.Results1. The shape of constructs was coordinate with the chambers at Week 2, but the constructs was surrounded by gel-like tissue. We observed adipogenesis and angiogenesis within the adipose tissue flaps, with the infiltration of inflammatory cells. At Week 4, the flaps had viable and well-shaped tissue and adipogenesis and angiogenesis stabilized. Between week 4 and week 8, the general view of constructs showed no significant change except for a capsule of fibrous connective tissue surrounding. HE stain showed typical mature adipose tissue with lobules structure.2.4 weeks after chamber removal, the volume of adipose tissue flap showed a sharp increasing, which is called two stage growth pattern. HE stain showed a similar result with that of the first stage growth process. Adipogenesis and angiogenesis were observed within the adipose tissue flaps again. The thickness of the capsule reduced. The whole mount staining showed the increasing numbers of Lectin+and Ki67+cells, which implied angiogenesis and proliferation further.3. After chamber removal, both of the two shapes constructs showed volume slight increasing and stabilized at week 24. It was no significant differences between week 16 and week 24. The constructs had a good shape maintenance and volume remained stable till week 24.DiscussionSeveral studies have sought to generate specifically-shaped vascularized and mature adipose tissues by embedding a vascularized pedicled fat flap in a chamber. The present study showed that large-volume vascularized adipose flaps that closely adopt the shape of their chambers can be generated in a rabbit model without having to use exogenous angiogenic or adipogenic growth factors or a biodegradable extracellular matrix support.The result showed that adipogenesis was evident in the chambered flaps at week 2 and that by week 8, the adipose tissue had matured. This reflects the fact that almost all destructive stimuli activate various intracellular and intercellular pathways, which then act in a coordinated fashion to restore tissue integrity and homeostasis. This wound-healing process is a broad and complex process that occurs in almost all kinds of tissue, including adipose tissue. Indeed, previous studies show that the trauma/inflammatory environment caused by surgery is a key factor that enhances the angiogenesis and adipogenesis of the adipose flap within the TEC. Moreover, the loss of cell-to-cell contact in the flap and the changes in the mechanical forces to which the flap is subject within the TEC may provide additional mitogenic stimuli by means of mechanotransduction. These mechanisms together are likely to have led to the rapid growth that we observed in the first 8 weeks after chamber implantation, as evidenced by the expansion in flap volume from 0.8 ml at the implantation to 5.09 (round-chambered flaps) and 3.65 (heart-shaped-chambered flaps) ml.However, the present study showed that after week 8, there was no further significant change in volume or histology in the chambered flaps. Increasing evidence suggests that implantation of any biomedical device into the body yields a characteristic wound-healing response. This response is initiated by a complex inflammatory response that involves multiple cell signaling pathways. It is then followed by the migration of fibroblasts to the surface of the implant and the subsequent differentiation of smooth muscle actin-expressing myofibroblasts. These cells deposit a collagen capsule that surrounds and effectively walls off the device from the surrounding soft tissue. The formation of this thick capsule enables the cells in the flap to regain cell-to-cell contact; this in turn limits the further growth of the adipose flap and tends to stabilize the wound-healing response. This explains why the adipogenesis and fibrosis balance plateaued 8 weeks after implantation in the present study, after which the chambered flap volume remained relatively constant right up until the end of the study at week 24.However, to our surprise, we found that when the chamber was removed at week 8, the capsule became thinner and the volume of the adipose flap again increased. The regrowth of the adipose tissue may be due to the up-regulation of trauma/inflammation environment caused by the chamber removal operation. The down-regulation of the foreign body reaction due to the removal of the chamber may also have reduced capsule formation, which would have further enhanced flap growth. However, after the initial spurt of regrowth seen at week 12, the growth started slowing down as the subcutaneous cavity became filled with new tissue. By week 24, growth had largely plateaued. Notably, although the capsule restricted flap growth in the chambered flaps, its continuing presence after chamber removal at 8 weeks caused the flap to maintain its original chamber-driven shape, even 16 weeks after chamber removal. This suggests that our approach could be useful for generating specifically-shaped adipose flaps that meet clinical demands. However, further studies that assess the long-term outcomes of this approach are warranted.Of particular interest was that the two operations at weeks 0 and 8 caused the flaps to gain large amounts of vascularized connective tissue, as shown by the sham-operated control rabbits. Despite this, there was no remarkable flap growth in these rabbits. Many studies show that changes in mechanical forces promote angiogenesis and the local elaboration and up-regulation of growth factors. Harunosuke et al. also found that external tissue suspension could induce adipose tissue enlargement by activating resident progenitor cells. Therefore, the poor flap growth in the control group may relate to the lack of mechanical effects after the inflammatory response that arose from the short-term trauma/inflammatory environment.Findlay et al. produced large volumes of tissue flap in the pig by using adipose tissue-engineering chambers. In their study, removing the chamber from the tissue-engineered flaps slowed down tissue growth relative to the weight growth rate of the animal. However, the present study showed the opposite happened in the rabbit model:the flap started growing again strongly as soon as the chamber was removed. This discrepancy may relate to the differences between the two animal models. Pigs grow continuously in both size and weight, unlike rabbits, whose growth plateaus at maturity. Thus, the continuous growth of the pigs may be a confounding factor in the study by Findlay et al. This possibility should be tested by examining other animal models.This is the first time to fabricate successfully pre-shaped large-volume engineered vascularized pedicled adipose flaps since Morrison’s group reported TEC technique. At the same time we put forward the concept of two stage growth pattern about TEC technique and observed the long-term stability for half year, which provide basis for reconstruction of soft tissue defects with TEC in future clinical experiment. Although the TEC strategy plan is not perfect and the constructs’shapes are not accurate with chambers, pre-shaped large-volume engineered vascularized pedicled adipose flaps with mature structure and long-term stability still have a wide indication for repairing different shapes large volume soft tissue defects in clinical. It can fabricate pre-shaped large-volume engineered vascularized pedicled adipose flap from small one in vivo, then transplant after expanding like skin expander technique. Consequently, it avoids donor deformity secondary to the traditional resection of fat flaps and is considered a safe, stable and effective way for clinical use.Conclusion1. The pre-shaped large-volume engineered vascularized pedicled adipose flaps can be induced by using tissue engineering chamber (TEC) technology.2. Two stage growth of adipose tissue flaps can achieve after removing the chambers, resulting in much larger volumes constructs.3. The shape and volume of adipose tissue flaps can remain relative stable for a long time after chambers removal.