Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering.

BACKGROUND: The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes. METHODS AND RESULTS: In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31-. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1+/CD31- vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31+ blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks. CONCLUSION: Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.

Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor-A expression and downregulation of miRNA-1, miRNA-206 and angiopoietin-1.

Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR-1 and miR-206 (p < 0.05) and angiopoietin-1 (p < 0.05) with upregulation of vascular endothelial growth factor-A (VEGF-A; p < 0.05). The miR-1 and angiopoietin-1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR-206 in L6 myoblasts caused a significant increase in VEGF-A expression (p < 0.05), further establishing that changes in VEGF-A expression are influenced by miR-206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel™ into isolated bilateral tissue engineering chambers incorporating a flow-through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin-1, miR-1 and miR-206. The relatively simple strategy of hypoxic preconditioning of implanted cells - including non-stem cell types - has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.

Composite breast reconstruction: Implant-based breast reconstruction with adjunctive lipofilling.

INTRODUCTION: Options for breast reconstructions enclose autologous tissue transfers or implants. Fat grafting is gaining more interest in this specific field of breast surgery. This study concentrates on the technique and aesthetic results of breast reconstruction with fat grafts combined with implants, in women who have undergone total mastectomy. METHODS: Breast reconstructions (n = 23) was performed using a protocol of intratissular expansion with serial deflation-lipofilling. In order to achieve the best aesthetic outcome, an additional small implant was placed. A retrospective data analysis was performed. In all patients a tissue expander was placed at the time of mastectomy or after removal of a previous breast reconstruction. The mean of lipoaspirate material for the reconstruction was 333 mL (range 120-715 mL). To create an adequate volume of the reconstructed breast, a supplementary small implant was placed, with a mean volume of 222 mL (range 125-375 mL). The mean follow-up was 33 months (range 19-50 months). RESULTS: A MRI analysis was performed in eight patients at least 9 months after the last lipofilling procedure, demonstrating a mean of 171 mL (range 64-538 mL) of transferred fat, a mean fat survival of 53% and a volume ratio of fat graft/implant of 0.97 (range 0,3-3,8). CONCLUSION: This composite technique of using autologous fat tissue and implants shows aesthetic pleasant results and must be considered as a valid alternative in a subset of patients. Further investigations to optimize the fat graft take must be encouraged.

Rigid removable cover for dorsal wound protection and tube fixation in pigs.

OBJECTIVE: To report the design and benefits of a rigid polyethylene cover 'shell' for the protection of dorsal torso wounds and tube fixation in pigs. METHODS: Open C-shaped polyethylene shells were designed to protect wounds and dressings on the dorsum of pigs used in research into negative pressure dressing-assisted wound healing. The shells were designed to resist trauma and contamination, to be comfortable and expansible, and to facilitate tube fixation and management. Strap fixation was optimised during experimentation. Efficacy was assessed by direct observation of dressing and wound protection, tube integrity and by macroscopic and microscopic assessments of wound healing. RESULTS: The shells effectively protected the wounds against blunt and sharp trauma, were simple to remove and reapply, were well tolerated and allowed for growth of the pigs. Circumferential neck straps attached by lateral straps to the shells proved critical. There was no wound infection or inflammation underlying the shells. Porting tubing via mid-dorsal holes in the shells and affixing the tubing just cranial to these holes prevented tube damage and traction, permitted tube management from outside the cages and allowed the pigs to move freely without becoming entangled. CONCLUSION: These shells effectively protected dorsal skin wounds and dressings, prevented tube damage and facilitated tube management in pigs. Similar systems may be useful for other production animals for wound management and for tube management with negative pressure wound healing, drain tubes or the delivery of nutrition, fluids or medications.

Diamond encapsulated photovoltaics for transdermal power delivery.

A safe, compact and robust means of wireless energy transfer across the skin barrier is a key requirement for implantable electronic devices. One possible approach is photovoltaic (PV) energy delivery using optical illumination at near infrared (NIR) wavelengths, to which the skin is highly transparent. In the work presented here, a subcutaneously implantable silicon PV cell, operated in conjunction with an external NIR laser diode, is developed as a power delivery system. The biocompatibility and long-term biostability of the implantable PV is ensured through the use of an hermetic container, comprising a transparent diamond capsule and platinum wire feedthroughs. A wavelength of 980 nm is identified as the optimum operating point based on the PV cell's external quantum efficiency, the skin's transmission spectrum, and the wavelength dependent safe exposure limit of the skin. In bench-top experiments using an external illumination intensity of 0.7 W/cm(2), a peak output power of 2.7 mW is delivered to the implant with an active PV cell dimension of 1.5 × 1.5 × 0.06 mm(3). This corresponds to a volumetric power output density of ~20 mW/mm(3), significantly higher than power densities achievable using inductively coupled coil-based approaches used in other medical implant systems. This approach paves the way for further ministration of bionic implants.

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers.

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

The influence of nitric oxide synthase 2 on cutaneous wound angiogenesis.

BACKGROUND: Inducible nitric oxide synthase (nitric oxide synthase 2, NOS 2) inhibition significantly suppresses chronically ischaemic skin flap survival, possibly because of reduced angiogenesis. OBJECTIVES: To investigate the effect of genetic NOS 2 inhibition on cutaneous wound angiogenesis in two in vivo murine models. The impact of NOS 2 manipulation on vascular endothelial growth factor (VEGF)-A stimulated and fibroblast growth factor (FGF)-2 stimulated angiogenesis was also investigated in the Matrigel(®) plug assay. METHODS: (i) Matrigel plugs/incisional wounds: two groups of NOS 2-/- mice and two groups of wild-type (WT) mice had bilateral Matrigel plugs containing 500 ng mL(-1) VEGF-A or 1000 ng mL(-1) FGF-2 injected subcutaneously in the abdomen. A 2·5 cm long dorsal incisional skin wound was created and sutured closed in the same animals. Wounds and plugs were explored at 7 or 12 days. (ii) Excisional wounds: dorsal 0·5 × 1·0 cm excisional skin wounds were created in four groups (two NOS 2-/- and two WT) and explored at 7 or 14 days. Wounds and Matrigel plugs were examined histologically and morphometrically for determination of percentage vascular volume (PVV). RESULTS: The PVV in NOS 2-/- incisional wounds and excisional wounds was significantly less than in WT wounds (P = 0·05 and P < 0·001, respectively). The PVV was significantly less in VEGF-A stimulated Matrigel plugs compared with FGF-2 stimulated plugs in NOS 2-/- mice (P < 0·01), but not in WT mice. CONCLUSIONS: NOS 2 is significantly involved in angiogenic signalling in healing skin wounds, particularly within the first 7 days. However, Matrigel plug vascularization suggests that the role of NOS 2 in angiogenesis is related to VEGF-A but not FGF-2 stimulated angiogenesis.

Pancreatic islet transplantation using vascularised chambers containing nerve growth factor ameliorates hyperglycaemia in diabetic mice.

Intraportal islet transplantation has shown initial promise for the treatment of type 1 diabetes. However, the portal vein site is associated with complications such as thrombosis and hepatic steatosis, leading to transplant failure. The aims of this study were to (1) test the feasibility of an alternative islet transplantation method that utilises a FDA-approved gelatin sponge as a novel islet carrier and (2) assess if exogenous addition of nerve growth factor (NGF) has any additional beneficial effects on graft performance in diabetic mice. Mice were rendered diabetic by a single intraperitoneal injection of streptozotocin. Five hundred syngeneic islets were seeded onto a Gelitaspon((R)) disc in the presence or absence of NGF, and placed into a silicone chamber surrounding the femoral neurovascular pedicle. Islet function was assessed by weekly monitoring of blood glucose levels and an intraperitoneal glucose tolerance test performed at the end of the study. Chambers were harvested for further histological analysis. Four of five mice transplanted with islets seeded onto Gelitaspon with NGF showed a significant reduction in blood glucose levels by 4 weeks after transplantation, and demonstrated a response similar to non-diabetic mice when tested with an intraperitoneal glucose tolerance test. Chamber tissue from this group contained islets with insulin-producing beta cells adjacent to the vascular pedicle. Islets seeded onto Gelitaspon with NGF and sited on femoral vessels using a tissue-engineering chamber offer an alternative method for islet transplantation in diabetic mice. This may have potential as a method for clinical islet transplantation.

Neovascularization in an arterio-venous loop-containing tissue engineering chamber: role of NADPH oxidase.

Using an in vivo arterio-venous loop-containing tissue-engineering chamber, we have created a variety of vascularized tissue blocks, including functional myocardium. The viability of the transplanted cells is limited by the rate of neovascularization in the chamber. A Nox2-containing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is thought to have a critical role in ischaemic angiogenesis. In this study we investigated whether NADPH oxidase is involved in the neovascularization process in the tissue-engineering chamber. New blood vessels originating from the venous and the arterial ends of the loop could be identified after 3 days, and the vessel density (by lectin staining) peaked after 7 days and was maintained for at least 14 days. This was accompanied by granulation tissue formation and concomitant increase in the mRNA level of Nox4 NADPH oxidase. Although the total level of Nox2 mRNA in the chamber tissue decreased from day 3 to day 7, immunohistochemistry identified a strong expression of Nox2 in the endothelial cells of the new vessels. In human microvascular endothelial cells, the NADPH oxidase inhibitor apocynin reduced NADPH oxidase activity and inhibited the angiogenic responses in vitro. Local treatment with the NADPH oxidase inhibitors apocynin or gp91ds-tat peptide significantly suppressed the vessel growth in the chamber. In conclusion, NADPH oxidase-dependent redox signalling is important for neovascularization in this novel tissue-engineering chamber in vivo, and boosting this signalling might be a new approach to extending vascularization and tissue growth.

Myogel, a novel, basement membrane-rich, extracellular matrix derived from skeletal muscle, is highly adipogenic in vivo and in vitro.

BACKGROUND/AIMS: Biological and synthetic scaffolds play important roles in tissue engineering and are being developed towards human clinical applications. Based on previous work from our laboratory, we propose that extracellular matrices from skeletal muscle could be developed for adipose tissue engineering. METHODS: Extracellular matrices (Myogels) extracted from skeletal muscle of various species were assessed using biochemical assays including ELISA and Western blotting. Biofunctionality was assessed using an in vitro differentiation assay and a tissue engineering construct model in the rat. RESULTS: Myogels were successfully extracted from mice, rats, pigs and humans. Myogels contained significant levels of laminin alpha4- and alpha2-subunits and collagen I compared to Matrigel, which contains laminin 1 (alpha1beta1gamma1) and collagen IV. Levels of growth factors such as fibroblast growth factor 2 were significantly higher than Matrigel, vascular endothelial growth factor-A levels were significantly lower and all other growth factors were comparable. Myogels reproducibly stimulated adipogenic differentiation of preadipocytes in vitro and the growth of adipose tissue in the rat. CONCLUSIONS: We found Myogel induces adipocyte differentiation in vitroand shows strong adipogenic potential in vivo, inducing the growth of well-vascularised adipose tissue. Myogel offers an alternative for current support scaffolds in adipose tissue engineering, allowing the scaling up of animal models towards clinical adipose tissue engineering applications.

Zymosan-induced inflammation stimulates neo-adipogenesis.

OBJECTIVE: To investigate the potential of inflammation to induce new adipose tissue formation in the in vivo environment. METHODS AND RESULTS: Using an established model of in vivo adipogenesis, a silicone chamber containing a Matrigel and fibroblast growth factor 2 (1 microg/ml) matrix was implanted into each groin of an adult male C57Bl6 mouse and vascularized with the inferior epigastric vessels. Sterile inflammation was induced in one of the two chambers by suspending Zymosan-A (ZA) (200-0.02 microg/ml) in the matrix at implantation. Adipose tissue formation was assessed at 6, 8, 12 and 24 weeks. ZA induced significant adipogenesis in an inverse dose-dependent manner (P<0.001). At 6 weeks adipose tissue formation was greatest with the lowest concentrations of ZA and least with the highest. Adipogenesis occurred both locally in the chamber containing ZA and in the ZA-free chamber in the contralateral groin of the same animal. ZA induced a systemic inflammatory response characterized by elevated serum tumour necrosis factor-alpha levels at early time points. Aminoguanidine (40 microg/ml) inhibited the adipogenic response to ZA-induced inflammation. Adipose tissue formed in response to ZA remained stable for 24 weeks, even when exposed to the normal tissue environment. CONCLUSIONS: These results demonstrate that inflammation can drive neo-adipogenesis in vivo. This suggests the existence of a positive feedback mechanism in obesity, whereby the state of chronic, low-grade inflammation, characteristic of the condition, may promote further adipogenesis. The mobilization and recruitment of a circulating population of adipose precursor cells is likely to be implicated in this mechanism.

Milrinone does not improve free flap survival in microvascular surgery.

Free flap microvascular surgery involves the transfer of a mobilised tissue flap with complete vascular re-anastomosis at the new site. Ischaemia frequently threatens flap survival and may require a return to the operating theatre for anastomotic revision. Arterial spasm and hypoperfusion are recognised as factors in flap ischaemia. Phosphodiesterase inhibitors such as milrinone may improve flap blood flow and possibly flap survival by arterial dilation and increasing cardiac output. To investigate the role of milrinone in this type of surgery, a double-blinded randomised controlled trial was conducted with 88 patients receiving either a milrinone bolus and infusion throughout surgery or placebo (normal saline). We found that milrinone did not improve graft survival, return to theatre rate, or surgically graded arterial spasm, but did require more vasopressor support. We conclude that intraoperative milrinone did not improve flap outcomes in microvascular surgery.

Erysipelas-like inflammation following breast surgery.

Impaired lymph drainage is an inevitable consequence of any form of surgery that disrupts lymphatics, resulting in a degree of lymphoedema that may vary from subtle to dramatic and although classically involving an entire limb, may be more localised, confined to only a small area such as a skin flap. Infection is a well-recognised complication of lymphoedema. However, not all inflammatory episodes occurring in the setting of lymphatic dysfunction can be clearly attributed to infection as this article demonstrates. Five patients presented over a 5-year period with distinctive erysipelas-like inflammation affecting the breast which occurred several weeks following reduction mammaplasty in four patients and breast reconstruction in one patient. No clinical response was obtained with standard antibiotics. This inflammatory problem may represent a previously unreported complication of breast surgery with an incidence of 4% following reduction mammaplasty. Recent research supports the notion that this type of episode is most likely to be due to a non-infective inflammatory process related to lymphatic dysfunction induced by surgery.

Vascularized tissue-engineered chambers promote survival and function of transplanted islets and improve glycemic control.

We have developed a chamber model of islet engraftment that optimizes islet survival by rapidly restoring islet-extracellular matrix relationships and vascularization. Our aim was to assess the ability of syngeneic adult islets seeded into blood vessel-containing chambers to correct streptozotocin-induced diabetes in mice. Approximately 350 syngeneic islets suspended in Matrigel extracellular matrix were inserted into chambers based on either the splenic or groin (epigastric) vascular beds, or, in the standard approach, injected under the renal capsule. Blood glucose was monitored weekly for 7 weeks, and an intraperitoneal glucose tolerance test performed at 6 weeks in the presence of the islet grafts. Relative to untreated diabetic animals, glycemic control significantly improved in all islet transplant groups, strongly correlating with islet counts in the graft (P<0.01), and with best results in the splenic chamber group. Glycemic control deteriorated after chambers were surgically removed at week 8. Immunohistochemistry revealed islets with abundant insulin content in grafts from all groups, but with significantly more islets in splenic chamber grafts than the other treatment groups (P<0.05). It is concluded that hyperglycemia in experimental type 1 diabetes can be effectively treated by islets seeded into a vascularized chamber functioning as a "pancreatic organoid."

The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber.

UNLABELLED: The distribution of hypoxic cells in an in vivo tissue engineering chamber was investigated up to 28 days post-implantation. METHODS: Arteriovenous loops were constructed and placed into bi-valved polycarbonate chambers containing 2 x 10(6) rat fibroblasts in basement membrane gel (BM gel). Chambers were inserted subcutaneously in the groin of male rats and harvested at 3 (n = 6), 7 (n = 6), 14 (n = 4) or 28 (n = 4) days. Ninety minutes before harvest, pimonidazole (60 mg/kg) was injected intraperitoneally. Chamber tissue was removed, immersion fixed, paraffin embedded, sectioned and stained immunohistochemically using hypoxyprobe-1 Mab that detects reduced pimonidazole adducts forming in cells, where pO2 < 10 mmHg. RESULTS: At 3 days a fibrin clot/BM gel framework filled the chamber. Seeded fibroblasts had largely died. The majority of 3 day chambers did not demonstrate tissue growth from the AV loop nor was pimonidazole binding present in these chambers. In one chamber in which tissue growth had occurred strong pimonidazole binding was evident within the new tissue. In four out of six 7 day chambers a broader proliferative zone existed extending up to 0.4 mm (approximately) from the AV loop endothelium which demonstrated intense pimonidazole binding. The two remaining 7 day chambers displayed even greater tissue growth (leading edge > 0.7 mm from the AV loop endothelium), but very weak or no pimonidazole binding. At 14 and 28 days the fibrin/BM gel matrix was replaced by mature vascularised connective tissue that did not bind pimonidazole. CONCLUSION: Employing a tissue engineering chamber, new tissue growth extending up to 0.4 mm from the AV loop endothelium (chambers < or = 7 days) demonstrated intense pimonidazole binding and, therefore, hypoxia. Tissue growth greater than 0.5 mm from the AV loop endothelium (7-28 days chambers) did not exhibit pimonidazole binding due to a significant increase in the number of new blood vessels and was, therefore, adequately oxygenated.

Comparing the efficacy of two fluorescent retrograde tracers in labeling the motor and sensory neuron populations of the rat sciatic nerve.

We compared the efficacy with which the fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) retrogradely label neutrons. Trace crystals were applied to the sciatic nerve exclusively (single label) or serially (double label). Unbiased cell counts showed that FB and DY label similar numbers of motoneurons (P=1.00, df 5) or DRG neurons (P=0.95, df 5) when applied exclusively. Plotting of motoneurons revealed a similar pattern of distribution of FB and DY labeled neurons. When the tracers were applied serially, 79% of labeled motoneurons and 77% of labeled DRG neurons were double-labeled irrespective of which tracer was applied first. Equal proportions of the remaining labeled neurons were single-labeled with FB or DY. These data show that FB and DY label equal numbers of motor and sensory neurons of the sciatic nerve following exclusive or serial application of tracers. These findings support the use of FB and DY together in serial fluorescent labeling experiments.

Vascularisation of tissue-engineered grafts: the regulation of angiogenesis in reconstructive surgery and in disease states.

Angiogenesis (the formation of new blood vessels) is essential for the growth of new tissue, tissue repair and wound healing. Tissue engineering, the construction of new tissue and organs for reparative purposes, relies on angiogenesis for the vascularisation of these new grafts. In tissue engineering, the emphasis to date has been on vascularisation of newly constructed tissue grafts by an extrinsic blood supply, and relatively little attention has been given to the possibility of building these grafts around an intrinsic blood supply. However, there are many disease processes, notably tumour growth, where excess angiogenesis can be a major problem. The purposes of this review are, first, to examine various methods of vascularising tissue-engineered grafts, and, second, to compare the role of angiogenesis in tissue engineering, where stimulation of angiogenesis is paramount, with pathological states, such as tumour growth, where angiogenesis needs to be inhibited.

Laser photoirradiation in digital flexor tendon repair.

This study evaluates tendon coaptation using Nd:YAG laser photoirradiation in an in vivo cockerel model. Using the intervinculum segments of the flexor profundus tendons, experimental transactions were performed. Tendon coaptation was then attempted using laser photoirradiation. Tendons were immediately examined for evidence of stable coaptation. After this assessment, specimens were excised and processed for electron microscopic examination and exposure to trypsin digestion. Despite varying multiple laser parameters, tissue welding was not observed. The subsequent functional and ultrastructural observations of irradiated tendon suggest that these changes are those of simple thermal denaturation. The results of this study suggest that when successful tissue welding has been observed in other tissue types, the mechanism is unlikely to be because of formation of intermolecular collagen bonds as hypothesized. An alternative hypothesis is that laser welding reflects photothermal coagulation of cytoplasmic peptides or nucleic acids liberated at the coaptation interface. This may explain the successful welding of cell-rich tissues such as bowel, vas deferens, and arteries and the observed failure of laser welding in collagen-rich but relatively hypocellular tendon.