Wayne A. Morrison

Cardiac Tissue Engineering in an In Vivo Vascularized Chamber

Background— Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. We have created vascularized pieces of beating cardiac muscle in the rat that are as thick as the adult rat right ventricle wall. Method and Results— Neonatal rat cardiomyocytes in Matrigel were implanted with an arteriovenous blood vessel loop into a 0.5-mL patented tissue-engineering chamber, located subcutaneously in the groin. Chambers were harvested 1, 4, and 10 weeks after insertion. At 4 and 10 weeks, all constructs that grew in the chambers contracted spontaneously. Immunostaining for &agr;-sarcomeric actin, troponin, and desmin showed that differentiated cardiomyocytes present in tissue at all time points formed a network of interconnected cells within a collagenous extracellular matrix. Constructs at 4 and 10 weeks were extensively vascularized. The maximum thickness of cardiac tissue generated was 1983 &mgr;m. Cardiomyocytes increased in size from 1 to 10 weeks and were positive for the proliferation markers Ki67 and PCNA. Connexin-43 stain indicated that gap junctions were present between cardiomyocytes at 4 and 10 weeks. Echocardiograms performed between 4 and 10 weeks showed that the tissue construct contracted spontaneously in vivo. In vitro organ bath experiments showed a typical cardiac muscle length-tension relationship, the ability to be paced from electrical field pulses up to 3 Hz, positive chronotropy to norepinephrine, and positive inotropy in response to calcium. Conclusion— In summary, the use of a vascularized tissue-engineering chamber allowed generation of a spontaneously beating 3-dimensional mass of cardiac tissue from neonatal rat cardiomyocytes. Further development of this vascularized model will increase the potential of cardiac tissue engineering to provide suitable replacement tissues for acquired and congenital defects.

New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials

The authors previously described a model of tissue engineering in rats that involves the insertion of a vascular pedicle and matrix material into a semirigid closed chamber, which is buried subcutaneously. The purpose of this study was to develop a comparable model in mice, which could enable genetic mutants to be used to more extensively study the mechanisms of the angiogenesis, matrix production, and cellular migration and differentiation that occur in these models. A model that involves placing a split silicone tube around blood vessels in the mouse groin was developed and was demonstrated to successfully induce the formation of new vascularized tissue. Two vessel configurations, namely, a flow-through pedicle (n = 18 for three time points) and a ligated vascular pedicle (n = 18), were compared. The suitability of chambers constructed from either polycarbonate or silicone and the effects of incorporating either Matrigel equivalent (n = 18) or poly(dl-lactic-co-glycolic acid) (n = 18) on angiogenesis and tissue production were also tested. Empty chambers, chambers with vessels only, and chambers with matrix only served as control chambers. The results demonstrated that a flow-through type of vascular pedicle, rather than a ligated pedicle, was more reliable in terms of patency, angiogenesis, and tissue production, as were silicone chambers, compared with polycarbonate chambers. Marked angiogenesis occurred with both types of extracellular matrix scaffolds, and there was evidence that native cells could migrate into and survive within the added matrix, generating a vascularized three-dimensional construct. When Matrigel was used as the matrix, the chambers filled with adipose tissue, creating a highly vascularized fat flap. In some cases, new breast-like acini and duct tissue appeared within the fat. When poly(dl-lactic-co-glycolic acid) was used, the chambers filled with granulation and fibrous tissue but no fat or breast tissue was observed. No significant amount of tissue was generated in the control chambers. Operative times were short (25 minutes), and two chambers could be inserted into each mouse. In summary, the authors have developed an in vivo murine model for studying angiogenesis and tissue-engineering applications that is technically simple and quick to establish, has a high patency rate, and is well tolerated by the animals.

An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct.

A major obstacle to 3‐dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the models vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.2±03.14% compared with 3.5±92.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.—Lokmic, Z., Stillaert, F., Morrison, W. A., Thompson, E. W., Mitchell, G. M. An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue‐engineered construct. FASEB J. 21, 511–522 (2007)

Formation of New Tissue from an Arteriovenous Loop in the Absence of Added Extracellular Matrix

A major requirement for the microsurgical repair of contour defects of the skin, for example, following removal of a skin cancer on the face, is a mass of vascularised subcutaneous tissue. Such tissue can be generated in vivo using basic tissue engineering principles. In previous studies in our laboratory, we have used a model comprising an arteriovenous (AV) shunt loop sandwiched in artificial dermis, placed in a cylindrical plastic growth chamber, and inserted subcutaneously to grow new connective tissue progressively up to 4 weeks. To learn more about the basic growth characteristics with this model, the same AV shunt loop within a chamber without added extracellular matrix was inserted subcutaneously into the groins of rats for 2, 4, or 12 weeks (n = 5 per group). There was a progressive increase in the mass and volume of tissue such that the chamber was two-thirds full after 12 weeks. Histological examination showed that at 2 weeks there was evidence of fibroblast and vascular outgrowth from the AV shunt, with the formation of granulation tissue, surrounded by a mass of coagulated exudate. At 4 weeks the connective tissue deposition was more extensive, with a mass of more mature granulation tissue containing considerable collagen. By 12 weeks there was an extensive, well vascularized mass of mature fibrous tissue. The blood vessels and residual adventitia of the AV shunt were the likely source of growth factors and of the cells which populated the chamber with new maturing connective tissue. A patent AV shunt in an isolated chamber appears to be the minimal requirement for the generation of new vascularized tissue that is potentially suitable for microsurgical transplantation.

Cross-facial nerve grafts and microneurovascular free muscle transfer for long established facial palsy.

Cross-facial nerve grafts followed in 4 to 12 months by microneurovascular free gracilis transplantation can produce adequate reconstruction in the lower two-thirds of a paralysed face. The mixed sensory and motor deep peroneal nerve and the small muscle bulk of the extensor digitorum brevis limit its usefulness in facial palsy. The gracilis has proved to be a much superior muscle. A feasible method for total reamination of unilateral facial palsy is presented.

The Influence of Extracellular Matrix on the Generation of Vascularized, Engineered, Transplantable Tissue

Abstract: In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no‐matrix control. Poly‐d,l‐lactic‐co‐glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Differentiation of human adipose-derived stem cells into beating cardiomyocytes

Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca2+ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.

Results of management of facial palsy with microvascular free-muscle transfer.

This paper reports our experience in facial reanimation using free innervated muscle transfer in 69 patients with long-term facial palsy. The majority of patients were treated in two stages with cross-facial nerve graft as the first stage and microvascular muscle transfer at the second stage. The gracilis muscle was used in 62 patients. A system of grading results has been utilized in the long-term evaluation. The overall final result was excellent or good in 51 percent of 47 patients who were available for follow-up. Although the results are not completely satisfactory, they justify the use of this approach to a difficult clinical problem. The results are improving as technical modifications to the procedure have evolved. The gracilis muscle is a reliable free transfer with internal anatomy conducive to use for reanimation of the paralyzed face. This type of transfer, in our experience, has proved superior to nonmicrosurgical methods for treatment of complete and severe incomplete facial palsy. The seventh cranial nerve is used in the innervation of the transferred muscle, the ipsilateral being preferable if available. The authors believe that use of the same cranial nerve is superior to methods that involve other cranial nerves, where spontaneity is often not achieved.

The late functional results of upper limb revascularization and replantation

The functional results in 25 of 30 patients after successful upper limb revascularization or replantation were evaluated by subjective-patient surveying and objective measurements. Young patients with complete, sharply amputated extremities at the wrist level or those with incomplete injuries and uninjured peripheral nerves had the best functional results. Multiple-level, diffuse crush, or avulsion injuries, even if the injuries were incomplete, and patients with high-level nerve injury had less return of function.

Tissue engineering skin flaps: which vascular carrier, arteriovenous shunt loop or arteriovenous bundle, has more potential for angiogenesis and tissue generation?

This study was designed to clarify which vascular carrier, the arteriovenous shunt loop or the arteriovenous bundle, has more potential as a vascular carrier for an artificial skin flap in rats. An arteriovenous shunt loop was constructed between the femoral artery and vein using an interpositional artery (group I) or vein (group II) graft. For arteriovenous bundle groups, the femoral artery and vein were used and subdivided into two groups: distal ligation type (group III) and flow-through type (group IV). The vascular pedicle was wrapped with an artificial dermis and implanted beneath the inguinal skin for 4 weeks. For the control group, a folded sheet of artificial dermis without any vascular carrier was embedded. In experiment 1, the volumes of generated tissue within the artificial dermis were measured in the experimental and control groups (n = 5 in each group). In experiment 2, the origin of new blood vessels sprouting from the arteriovenous shunt loop and arteriovenous bundle were evaluated histologically. The volume of generated tissue in the shunt groups was significantly greater than that in the bundle groups (p < 0.01). However, the bundle groups also showed a great potential for producing new tissue. Serial histological studies showed that new capillaries were derived not only from the vasa vasorum of the femoral vessels but directly from the femoral vein in both the shunt and the bundle groups. This “sprouting” was extensively exhibited in the group III. Although the arteriovenous shunt loop showed a greater potential for producing new tissue and capillaries, the distal ligation type of bundle was thought to be an effective and practical vascular carrier for producing a tissue-engineered skin flap.