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Background: Current clinical approaches to regenerate temporomandibular joint (TMJ) articulating cartilage defects only treat the symptoms (i.e. pain and dysfunction) and do not seek to restore joint integrity for long-term relief. Therefore, we investigated a novel self-assembling tissue-engineered cartilage to overcome this significant clinical issue for TMJ regenerative purposes., Objectives: Examine the maturation of dynamic self-regenerating cartilage (dSRC) using auricular chondrocytes and evaluate a novel combinatorial approach with fractional laser treatment and dSRC implantation for TMJ cartilage repair., Materials and Methods: A suspension of 10 7 freshly harvested rabbit ear chondrocytes was cultured under a continuous reciprocating motion to form the dSRC. After 2, 4 and 8 weeks of culture, dSRC samples were stained with H&E, Safranin-O and Toluidine Blue. Immunohistochemistry (IHC) was performed for collagens type I and II. Channels (300-500 μm diameter and 1.2-1.5 mm depth) were created in six freshly harvested condyles using a fractional Erbium laser. Two groups were tested: dSRC in a laser-ablated lesion (experimental) and an empty laser-ablated channel (control). TMJ condyles were cultured for up to 8 weeks and analysed as described above., Results: H&E staining showed a high cell density in dSRC compared to native cartilage. All dSRC groups demonstrated intense Safranin-O staining, indicating high glycosaminoglycan (GAG) production and intense Toluidine Blue staining showed high proteoglycan content. IHC confirmed that dSRC consisted predominantly of collagen type II. The experimental group showed improved cartilage repair at both time points compared to the empty channels., Conclusion: dSRC viability and successful matrix formation were demonstrated in vitro. The combination of fractional laser ablation and dSRC implantation enhanced cartilage repair., (© 2024 John Wiley & Sons Ltd.)

Photochemical sealing of a nerve wrap over the repair site isolates and optimizes the regenerating nerve microenvironment. To facilitate clinical adoption of the technology, we investigated photosealed autologous tissue in a rodent sciatic nerve transection and repair model. Rats underwent transection of the sciatic nerve with repair performed in three groups: standard microsurgical neurorrhaphy (SN) and photochemical sealing with a crosslinked human amnion (xHAM) or autologous vein. Functional recovery was assessed at four-week intervals using footprint analysis. Gastrocnemius muscle mass preservation, histology, and nerve histomorphometry were evaluated at 120 days. Nerves treated with a PTB-sealed autologous vein improved functional recovery at 120 days although the comparison between groups was not significantly different (SN: -58.4 +/- 10.9; XHAM: -57.9 +/- 8.7; Vein: -52.4 +/- 17.1). Good muscle mass preservation was observed in all groups, with no statistical differences between groups (SN: 69 +/- 7%; XHAM: 70 +/- 7%; Vein: 70 +/- 7%). Histomorphometry showed good axonal regeneration in all repair techniques. These results demonstrate that peripheral nerve repair using photosealed autologous veins produced regeneration at least equivalent to current gold-standard microsurgery. The use of autologous veins removes costs and foreign body concerns and would be readily available during surgery. This study illustrates a new repair method that could restore normal endoneurial homeostasis with minimal trauma following severe nerve injury.

Objectives: This study investigated a novel strategy for improving regenerative cartilage outcomes. It combines fractional laser treatment with the implantation of neocartilage generated from autologous dynamic Self-Regenerating Cartilage (dSRC)., Methods: dSRC was generated in vitro from harvested autologous swine chondrocytes. Culture was performed for 2, 4, 8, 10, and 12 weeks to study matrix maturation. Matrix formation and implant integration were also studied in vitro in swine cartilage discs using dSRC or cultured chondrocytes injected into CO 2 laser-ablated or mechanically punched holes. Cartilage discs were cultured for up to 8 weeks, harvested, and evaluated histologically and immunohistochemically., Results: The dSRC matrix was injectable by week 2, and matrices grew larger and more solid with time, generating a contiguous neocartilage matrix by week 8. Hypercellular density in dSRC at week 2 decreased over time and approached that of native cartilage by week 8. All dSRC groups exhibited high glycosaminoglycan (GAG) production, and immunohistochemical staining confirmed that the matrix was typical of normal hyaline cartilage, being rich in collagen type II. After 8 weeks in cartilage lesions in vitro, dSRC constructs generated a contiguous cartilage matrix, while isolated cultured chondrocytes exhibited only a sparse pericellular matrix. dSRC-treated lesions exhibited high GAG production compared to those treated with isolated chondrocytes., Conclusions: Isolated dSRC exhibits hyaline cartilage formation, matures over time, and generates contiguous articular cartilage matrix in fractional laser-created microenvironments in vitro, being well integrated with native cartilage.

Background: In the last decade, tissue-engineering strategies for regenerating the temporomandibular joint (TMJ) have been investigated. This may be a promising strategy for the minimally invasive restoration of joint integrity., Objectives: To evaluate whether dental pulp stem cells (DPSCs) loaded in a light-occured hydrogel made of gelatin methacryloyl (GelMA) enhance the regeneration of osteochondral defects in the rabbit TMJ., Materials and Methods: Defects were filled with GelMA alone (control group; n = 4) or filled with GelMA loaded with rabbit DPSCs (experimental group; n = 4), In one group, the TMJ capsule was opened without creating a defect (sham group; n = 2). The following micro-CT parameters were analysed: bone volume to total volume ratio (BV/TV%) and bone mineral density (BMD). Histological evaluation was performed to assess cartilage regeneration features. A semi-quantitative scoring system was also used to evaluate the defects., Results: All groups had no statistical difference regarding the micro-CT parameters. The highest mean healing score was found for the experimental group. After 4 weeks, there were no signs of hydrogel in either group or no signs of inflammation in the adjacent tissues. The tissue formed in the defect was dense fibrous connective tissue., Conclusion: Adding DPSCs to GelMA did not provide a regenerative enhancement in TMJ osteochondral defects. This resulted in similar micro-CT parameters after 4 weeks of healing, with improved signs of subchondral bone regeneration but no cartilage regeneration., (© 2023 John Wiley & Sons Ltd.)

Critical-sized bone defects (CSBDs) represent a significant clinical challenge, stimulating researchers to seek new methods for successful bone reconstruction. The aim of this systematic review is to assess whether bone marrow stem cells (BMSCs) combined with tissue-engineered scaffolds have demonstrated improved bone regeneration in the treatment of CSBD in large preclinical animal models. A search of electronic databases (PubMed, Embase, Web of Science, and Cochrane Library) focused on in vivo large animal studies identified 10 articles according to the following inclusion criteria: (1) in vivo large animal models with segmental bone defects; (2) treatment with tissue-engineered scaffolds combined with BMSCs; (3) the presence of a control group; and (4) a minimum of a histological analysis outcome. Animal research: reporting of in Vivo Experiments guidelines were used for quality assessment, and Systematic Review Center for Laboratory animal Experimentation's risk of bias tool was used to define internal validity. The results demonstrated that tissue-engineered scaffolds, either from autografts or allografts, when combined with BMSCs provide improved bone mineralization and bone formation, including a critical role in the remodeling phase of bone healing. BMSC-seeded scaffolds showed improved biomechanical properties and microarchitecture properties of the regenerated bone when compared with untreated and scaffold-alone groups. This review highlights the efficacy of tissue engineering strategies for the repair of extensive bone defects in preclinical large-animal models. In particular, the use of mesenchymal stem cells, combined with bioscaffolds, seems to be a successful method in comparison to cell-free scaffolds.

Purpose: Photosealing of many biological tissues can be achieved using a biocompatible material in combination with a dye that is activated by visible light to chemically bond over the tissue defect via protein cross-linking reactions. The aim of this study was to test the efficacy of photosealing using a commercially available biomembrane (AmnioExcel Plus) to securely close dural defects in comparison to another sutureless method (fibrin glue) in terms of repair strength., Methods: Two-millimeter diameter holes were created in dura harvested from New Zealand white rabbits and repaired ex vivo using one of two methods: (1) in n = 10 samples, photosealing was used to bond a 6-mm-diameter AmnioExcel Plus patch over the dural defect, and (2) in n = 10 samples, fibrin glue was used to attach the same patch over the dural defect. Repaired dura samples were then subjected to burst pressure testing. Histological analysis was also performed of photosealed dura., Results: The mean burst pressures of rabbit dura repaired with photosealing and fibrin glue were 302 ± 149 mmHg and 26 ± 24 mmHg, respectively. The increased repair strength using photosealing was statistically significant and considerably higher than the normal intracranial pressure of ~ 20 mmHg. Histology demonstrated a tight union at the interface between the dura surface and patch with no disruption of the dura structure., Conclusion: The results of this study suggest that photosealing performs better than fibrin glue for the fixation of a patch for ex vivo repair of small dural defects. Photosealing is worthy of testing in pre-clinical models for the repair of dural defects., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background . There have been few advances in technique since vascular anastomosis was performed with silk suture on a curved needle in 1902. This technique results in disruption of the endothelium with exposed intraluminal suture, both of which may lead to thrombocyte aggregation, intimal hyperplasia, and vascular stenosis. A variety of alternative techniques have been explored, with limited success. Photochemical tissue bonding (PTB) is a light-activated methodology of rapidly cross-linking tissue interfaces at the molecular level. Herein, we describe a new technique for anastomosis of venous interposition graft in an ovine model of femoral artery bypass utilizing PTB. Methods . Polypay specific pathogen free sheep (n = 5; 40-45 kg) underwent femoral artery bypass utilizing saphenous vein. The femoral artery was transected and reversed saphenous vein was implanted as an interposition graft. The proximal anastomosis was created as a vein-over-artery cuff utilizing PTB, and the distal anastomosis was created with standard interrupted 8-0 polypropylene suture. Four weeks post-index operation, femoral angiogram was performed to evaluate patency, tortuosity, and luminal diameter. All bypass grafts were harvested and longitudinal and transverse histological sections from the proximal anastomosis were analyzed. Results . The PTB anastomoses (n = 5) were immediately watertight and patent. All animals survived the 28-day study duration. Angiography revealed patent grafts with no aneurysm or stenosis (n = 5). Histologic examination revealed integration of the venous endothelium with the arterial adventitia. Conclusion . Photochemical tissue bonding creates an immediate strong, watertight vascular anastomosis that can withstand physiologic arterial pressure and remains patent at 28 days without the need for intraluminal suture.

Painful neuroma is a frequent sequela of peripheral nerve injury which can result in pain and decreased quality of life for the patient, often necessitating surgical intervention. End neuromas are benign neural tumors that commonly form after nerve transection, when axons from the proximal nerve stump regenerate in a disorganized manner in an attempt to recreate nerve continuity. Inflammation and collagen remodeling leads to a bulbous end neuroma which can become symptomatic and result in decreased quality of life. This review covers surgical prophylaxis of end neuroma formation at time of injury, rather than treatment of existing neuroma and prevention of recurrence. The current accepted methods to prevent end neuroma formation at time of injury include different mechanisms to inhibit the regenerative response or provide a conduit for organized regrowth, with mixed results. Approaches include proximal nerve stump capping, nerve implantation into bone, muscle and vein, various pharmacologic methods to inhibit axonal growth, and mechanisms to guide axonal growth after injury. This article reviews historical treatments that aimed to prevent end neuroma formation as well as current and experimental treatments, and seeks to provide a concise, comprehensive resource for current and future therapies aimed at preventing neuroma formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Scott, Winograd and Redmond.)

Neuroma is a common sequela of traumatic peripheral nerve injury that can result in pain and decreased quality of life for patients. Neuromas result from axonal outgrowth in an attempt to reestablish continuity with the disrupted distal nerve end. Photosealing is a light-activated technique whereby tissues can be securely isolated in a strong and secure manner. This study investigated whether photosealing of autologous vein and crosslinked human amniotic membrane (xHAM) to cap the proximal stump of transected sciatic nerve would prevent disorganized axonal regeneration and neuroma in a rat model., Methods: The right sciatic nerve of Lewis rats (n = 27, 300-350 g) was transected 1 cm proximal to the trifurcation. Animals were randomized to one of three groups (n = 9): no further intervention (Group 1), photosealing with xHAM (Group 2), or photosealing with vein (Group 3). After 60 days, rats were euthanized and their right hindlimbs were re-explored for evidence of disorganized axonal regeneration and/or bulbous neuroma., Results: All untreated control animals were found to have protruding nerve fibers, often invading the adjacent muscle, and 33% of these control animals exhibited a bulbous neuroma. Photosealing with xHAM successfully capped 100% of nerves, with no observable axonal outgrowth. Photosealing with vein prevented axonal outgrowth in eight of nine nerves. No bulbous neuroma was found in any photosealed nerves., Conclusion: Nerve capping with photosealed xHAM or autologous vein can prevent axonal outgrowth in transected nerves, therefore decreasing the likelihood of symptomatic neuroma formation following nerve transection injury or surgical intervention., (Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Background: The autologous vein remains the standard conduit for lower extremity and coronary artery bypass grafting despite a 30%-50% 5-y failure rate, primarily attributable to intimal hyperplasia (IH) that develops in the midterm period (3-24 mo) of graft maturation. Our group discovered that externally strengthening vein grafts by cross-linking the adventitial collagen with photochemical tissue passivation (PTP) mitigates IH in an arteriovenous model at 4 wk. We now investigate whether this effect is retained in the midterm period follow-up., Methods: Six Hanford miniature pigs received bilateral carotid artery interposition vein grafts. In each animal, the external surface of one graft was treated with PTP before grafting, whereas the opposite side served as the untreated control. The grafts were harvested after 3 mo. Ultrasound evaluation of all vein grafts was performed at the time of grafting and harvest. The grafts were also evaluated histomorphometrically and immunohistologically for markers of IH., Results: All vein grafts were patent at 3 mo except one graft in the PTP-treated group because of early technical failure. The control vein grafts had significantly greater IH than PTP-treated grafts at 3 mo, as evidenced by the intimal area (2.6 ± 1.0 mm 2 versus 1.4 ± 1.5 mm 2 , respectively, P = 0.045) and medial area (5.1 ± 1.9 mm 2 versus 2.7 ± 2.4 mm 2 , respectively, P = 0.048). The control grafts had an increased presence and proliferation of mural myofibroblasts with greater smooth muscle actin and proliferating cell nuclear antigen staining., Conclusions: PTP treatment to the external surface of the vein grafts decreases IH at 3 mo after arteriovenous grafting and may prevent future graft failure., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Wound contracture formation from excessive myofibroblast activity can result in debilitating morbidities. There are currently no treatments to prevent contracture. Photochemical tissue passivation (PTP), an established, safe, and user-friendly treatment modality, crosslinks collagen by a light-activated process, thus modulating the wound healing response and scarring. We hypothesised that PTP treatment would reinforce wounds by blunting the fibrotic response thus limiting contracture., Study Design/materials and Methods: Full-thickness, 1 cm × 1 cm excisional wounds were created on the dorsum of 32 C57BL/6 mice. Treated wounds were painted with photosensitizing dye and exposed to visible light. Wounds were serially photographed over 6 weeks to measure wound contracture. At 7, 14, 21, and 42 days after wound creation, mice were euthanized and wounds were harvested for histologic review by a dermatopathologist., Results: By Day 7, control wounds had significantly more contracture than those treated with PTP (33.0 ± 17.1% and 19.3 ± 9.0%, respectively; P = 0.011). PTP-treated wounds maintained approximately 20% less contracture than controls from Day 14 and on (P < 0.05). By Day 42, wounds had contracted by 86.9 ± 5.5% in controls and 64.2 ± 3.2% in PTP-treated wounds (P < 0.03). Histologically, PTP wounds had earlier growth and development of dermal collagen, neovascularization, and development of skin appendages, compared with control wounds., Conclusions: PTP significantly limits contracture of full-thickness wounds and improves wound healing. PTP-treated wounds histologically demonstrate more mature structural organization than untreated wounds and closely resemble native skin. PTP treatment may be applicable not only for excisional wounds, but also for wounds with a high incidence of contracture and associated morbidity. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

This review summarizes research on many of the potential applications of photosensitized crosslinking of tissue proteins in surgery and current knowledge of the photochemical mechanisms underlying formation of the covalent protein-protein crosslinks involved. Initially developed to close wounds or reattach tissues, protein photocrosslinking has also been demonstrated to stiffen and strengthen tissues, decrease inflammatory responses and facilitate tissue bioengineering. These treatments appear to result largely from crosslinks within and between collagen molecules in tissue that typically form by an oxygen-dependent mechanism. Surgical applications discussed include sealing wounds in skin, cornea and bowel; reattaching severed nerves, blood vessels and tendons; strengthening cornea and vein; reducing capsular contracture after breast implants; and regenerating joint cartilage., (© 2019 American Society for Photobiology.)

Severe peripheral nerve injuries often result in partial repair and lifelong disabilities in patients. New surgical techniques and better graft tissues are being studied to accelerate regeneration and improve functional recovery. Currently, limited tools are available to provide in vivo monitoring of changes in nerve physiology such as myelination and vascularization, and this has impeded the development of new therapeutic options. We have developed a wide-field and label-free functional microscopy platform based on angiographic and vectorial birefringence methods in optical coherence tomography (OCT). By incorporating the directionality of the birefringence, which was neglected in the previously reported polarization-sensitive OCT techniques for nerve imaging, vectorial birefringence contrast reveals internal nerve microanatomy and allows for quantification of local myelination with superior sensitivity. Advanced OCT angiography is applied in parallel to image the three-dimensional vascular networks within the nerve over wide-fields. Furthermore, by combining vectorial birefringence and angiography, intraneural vessels can be discriminated from those of the surrounding tissues. The technique is used to provide longitudinal imaging of myelination and revascularization in the rodent sciatic nerve model, i.e. imaged at certain sequential time-points during regeneration. The animals were exposed to either crush or transection injuries, and in the case of transection, were repaired using an autologous nerve graft or acellular nerve allograft. Such label-free functional imaging by the platform can provide new insights into the mechanisms that limit regeneration and functional recovery, and may ultimately provide intraoperative assessment in human subjects.

Objective: We hypothesized that decreasing vein compliance would protect the vein against stretch injury and reduce intimal hyperplasia (IH)., Background: Although arteriovenous fistulas (AVFs) are the criterion standard for vascular access, their effectiveness is limited by poor patency with 40% to 60% failing due to IH. Venous stretch injury from exposure to arterial pressure induces IH. Photochemical tissue passivation (PTP) crosslinks adventitial collagen, decreasing vein compliance to resemble that of an artery., Methods: AVFs were created between the femoral artery and epigastric vein in rats (n = 29). PTP was performed on the vein immediately before vessel anastomosis. AVFs were harvested after four weeks. Venous diameter was measured at the initial procedure and harvest. Intimal area was measured for each segment. Ultrasound was performed at harvest to measure AVF flow., Results: Following AVF construction, venous diameter increased by 10% ± 18% for PTP-treated vessels and 78% ± 27% for controls (P ≤ 0.0001). At one month, PTP reduced AVF dilation by 71% compared to control (69% ± 29% vs 241% ± 78%; P ≤ 0.0001). Both juxta-anastomotic intimal area and total intimal area were reduced in PTP-treated vessels compared to control vessels. Specifically, intimal area was 0.024 ± 0.018 and 0.095 ± 0.089 mm for PTP-treated juxta-anastomotic segments of AVF and control, respectively (P < 0.05). Mean total intimal area for PTP-treated and control AVF were 0.080 ± 0.042 and 0.190 ± 0.110 mm, respectively (P < 0.03). AVF flow was 46.9 ± 35.3 and 19.1 ± 10.1 mL/min for PTP-treated and control AVF, respectively (P < 0.109)., Conclusions: These data demonstrate that PTP represents a promising therapy for the prevention of AVF IH, a process that might improve surgical outcomes for patients receiving hemodialysis.

Background: Photochemical tissue bonding (PTB) is a sutureless, light-activated technique that produces a watertight, microvascular repair with minimal inflammation compared to standard microsurgery. However, it is practically limited by the need for a clinically viable luminal support system. The aim of this study was to evaluate a hollow biocompatible stent to provide adequate luminal support to facilitate vascular anastomosis using the PTB technique., Methods: Forty rats underwent unilateral femoral artery transection. Five rats were used to optimize the stent delivery method, and the remaining 35 rats were randomized into three groups: (1) standard suture repair with 10-0 nylon microsuture (SR), (2) standard suture repair over the stent (SR + S), or (3) PTB repair over stent (PTB + S). For the PTB group, a 2-mm overlapping cuff was painted with 0.1% (wt/vol) Rose Bengal then illuminated for 30 seconds on each side (532 nm, 0.5 W/cm, 30 J/cm). Anastomotic leak and vessel patency (immediate, 1 hour, and 1 week postoperatively) were assessed., Results: Vessels in all three groups were patent immediately and at 1 hour postoperatively. After 1 week, all animals displayed patency in the SR group, while only 5 of 14 and 2 of 8 surviving animals had patent vessels in the PTB + S and SR + S groups, respectively., Conclusions: This study demonstrated successful use of an intraluminal stent for acute microvascular anastomosis using the PTB technique. However, the longer-term presence of the stent at the anastomotic site led to thrombosis in multiple cases. A rapidly dissolvable stent should facilitate a light-activated microvascular anastomosis with excellent long-term patency.

Objective: Saphenous vein is the conduit of choice for bypass grafting. Saphenous vein grafts have poor long-term patency rates because of intimal hyperplasia (IH) and subsequent accelerated atherosclerosis. One of the primary triggers of IH is endothelial injury resulting from excessive dilation of the vein after exposure to arterial pressures. Photochemical tissue passivation (PTP) is a technology that cross-links adventitial collagen by a light-activated process, which limits dilation by improving vessel compliance. The objective of this study was to investigate whether PTP limits the development of IH in a rodent venous interposition graft model., Methods: PTP is accomplished by coating venous adventitia with a photosensitizing dye and exposing it to light. To assess the degree of collagen cross-linking after PTP treatment, a biodegradation assay was performed. Venous interposition grafts were placed in the femoral artery of Sprague-Dawley rats. Rats were euthanized after 4 weeks, and intimal thickness was measured histologically. Vein dilation at the time of the initial procedure was also measured., Results: Time to digestion was 63 ± 7 minutes for controls, 101 ± 2.4 minutes for rose bengal (RB), and 300 ± 0 minutes for PTP (P < .001 PTP vs control). A total of 37 animals underwent the procedure: 12 PTP, 12 RB only, and 13 untreated controls. Dilation of the graft after clamp release was 99% for control, 65% for RB only, and 19% for PTP-treated (P < .001 PTP vs control). Intimal thickness was 77 ± 59 μm in controls, 60 ± 27 μm in RB only, and 33 ± 28 μm in PTP-treated grafts. There was a statistically significant 57% reduction in intimal thickness after treatment with PTP compared with untreated controls (P = .03)., Conclusions: PTP treatment of venous interposition grafts in a rat model resulted in significant collagen cross-linking, decreased vessel compliance, and significant reduction in IH., (Copyright © 2015 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Background: The study was aimed to compare the effects of small intestinal submucosa (SIS) and human amniotic membrane (HAM) on Achilles tendon healing., Methods: A total of 48 New Zealand white rabbits were divided into two groups. A full-thickness transverse tenotomy was made at the right leg of the rabbits. Then, the laceration site was wrapped with HAM (P/A group) or SIS (P/S group). The ultimate stress (US) and Young's modulus (E) of the tendons were detected for biomechanical analysis. Histological evaluation was performed using hematoxylin and eosin, immunohistochemical, and immunofluorescent stain. Expression of collagen I was detected by western blot analysis, and levels of inflammatory cytokines IL-1β, IL-6, and TNF-α were measured. Finally, adhesion formation was evaluated., Results: There were no significant differences in filamentous adhesion, cross-sectional areas of the laceration sites, levels of inflammatory response, and collagen type I expression between the P/A and P/S groups (p > 0.05). Compared with the P/A group, the US and E values were significantly higher in the P/S group at day 7 (p < 0.05) and at day 14 (p < 0.05). In addition, vascularity was significantly higher in the P/S group than that in the P/A group at day 3 (p < 0.05), day 7 (p < 0.01), and day 9 (p < 0.05)., Conclusions: SIS showed superior biomechanical properties and neovascularization over HAM in treatment of Achilles tendon injury in the early stage of healing.

Background: Bypass grafting remains the standard of care for coronary artery disease and severe lower extremity ischemia. Efficacy is limited by poor long-term venous graft patency secondary to intimal hyperplasia (IH) caused by venous injury upon exposure to arterial pressure. We investigate whether photochemical tissue passivation (PTP) treatment of vein grafts modulates smooth muscle cell (SMC) proliferation and migration, and inhibits development of IH., Methods and Results: PTP was performed at increasing fluences up to 120 J/cm(2) on porcine veins. Tensiometry performed to assess vessel elasticity/stiffness showed increased stiffness with increasing fluence until plateauing at 90 J/cm(2) (median, interquartile range [IQR]). At 90 J/cm(2), PTP-treated vessels had a 10-fold greater Young's modulus than untreated controls (954 [IQR, 2217] vs 99 kPa [IQR, 63]; P=0.03). Each pig received a PTP-treated and untreated carotid artery venous interposition graft. At 4-weeks, intimal/medial areas were assessed. PTP reduced the degree of IH by 66% and medial hypertrophy by 49%. Intimal area was 3.91 (IQR, 1.2) and 1.3 mm(2) (IQR, 0.97; P≤0.001) in untreated and PTP-treated grafts, respectively. Medial area was 9.2 (IQR, 3.2) and 4.7 mm(2) (IQR, 2.0; P≤0.001) in untreated and PTP-treated grafts, respectively. Immunohistochemistry was performed to assess alpha-smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA). Objectively, there were less SMA-positive cells within the intima/media of PTP-treated vessels than controls. There was an increase in PCNA-positive cells within control vein grafts (18% [IQR, 5.3]) versus PTP-treated vein grafts (5% [IQR, 0.9]; P=0.02)., Conclusions: By strengthening vein grafts, PTP decreases SMC proliferation and migration, thereby reducing IH., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Injuries to the articular cartilage surface are challenging to repair because cartilage possesses a limited capacity for self-repair. The outcomes of current clinical procedures aimed to address these injuries are inconsistent and unsatisfactory. We have developed a novel method for generating hyaline articular cartilage to improve the outcome of joint surface repair. A suspension of 10(7) swine chondrocytes was cultured under reciprocating motion for 14 days. The resulting dynamic self-regenerating cartilage (dSRC) was placed in a cartilage ring and capped with fibrin and collagen gel. A control group consisted of chondrocytes encapsulated in fibrin gel. Constructs were implanted subcutaneously in nude mice and harvested after 6 weeks. Gross, histological, immunohistochemical, biochemical, and biomechanical analyses were performed. In swine patellar groove, dSRC was implanted into osteochondral defects capped with collagen gel and compared to defects filled with osteochondral plugs, collagen gel, or left empty after 6 weeks. In mice, the fibrin- and collagen-capped dSRC constructs showed enhanced contiguous cartilage matrix formation over the control of cells encapsulated in fibrin gel. Biochemically, the fibrin and collagen gel dSRC groups were statistically improved in glycosaminoglycan and hydroxyproline content compared to the control. There was no statistical difference in the biomechanical data between the dSRC groups and the control. The swine model also showed contiguous cartilage matrix in the dSRC group but not in the collagen gel and empty defects. These data demonstrate the survivability and successful matrix formation of dSRC under the mechanical forces experienced by normal hyaline cartilage in the knee joint. The results from this study demonstrate that dSRC capped with hydrogels successfully engineers contiguous articular cartilage matrix in both nonload-bearing and load-bearing environments.

Background and Objective: Colonic anastomotic failure is a dreaded complication, and multiple surgical techniques have failed to eliminate it. Photochemical tissue bonding (PTB) is a method of sealing tissue surfaces by light-activated crosslinking. We evaluated if a human amniotic membrane (HAM), sealed over the anastomotic line by PTB, increases the anastomotic strength., Study Design: Sprague-Dawley rats underwent midline laparotomy followed by surgical transection of the left colon. Animals were randomized to colonic anastomosis by one of the following methods (20 per group): single-layer continuous circumferential suture repair (SR); SR with a HAM wrap attached by suture (SR+ HAM-S); SR with HAM bonded photochemically over the anastomotic site using 532 nm light (SR+ HAM-PTB); approximation of the bowel ends with only three sutures and sealing with HAM-PTB (3+ HAM-PTB). A control group underwent laparotomy alone with no colon resection (NR). Sub-groups (n = 10) were sacrificed at days 3 and 7 post-operatively and adhesions were evaluated. A 6 cm section of colon was then removed and strength of anastomosis evaluated by burst pressure (BP) measurement., Results: A fourfold increase in BP was observed in the SR+ HAM-PTB group compared to suture repair alone (94 ± 3 vs. 25 ± 8 mm Hg, P < 0.0001) at day 3. At day 7 the burst pressures were 165 ± 40 and 145 ± 31 mm Hg (P = 1), respectively. A significant decrease in peri-anastomotic adhesions was observed in the SR+ HAM-PTB group compared to the SR group at both time points (P < 0.001)., Conclusion: Sealing sutured colonic anastomotic lines with HAM-PTB increases the early strength of the repair and reduces peri-anastomotic adhesions. Lasers Surg. Med. 48:530-537, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Introduction Photochemical tissue bonding (PTB) uses visible light to create sutureless, watertight bonds between two apposed tissue surfaces stained with photoactive dye. In phase 1 of this two-phase study, nerve gaps repaired with bonded isografts were superior to sutured isografts. When autograft demand exceeds supply, acellular nerve allograft (ANA) is an alternative although outcomes are typically inferior. This study assesses the efficacy of PTB when used with ANA. Methods Overall 20 male Lewis rats had 15-mm left sciatic nerve gaps repaired using ANA. ANAs were secured using epineurial suture (group 1) or PTB (group 2). Outcomes were assessed using sciatic function index (SFI), gastrocnemius muscle mass retention, and nerve histomorphometry. Historical controls from phase 1 were used to compare the performance of ANA with isograft. Statistical analysis was performed using analysis of variance and Bonferroni all-pairs comparison. Results All ANAs had signs of successful regeneration. Mean values for SFI, muscle mass retention, nerve fiber diameter, axon diameter, and myelin thickness were not significantly different between ANA + suture and ANA + PTB. On comparative analysis, ANA + suture performed significantly worse than isograft + suture from phase 1. However, ANA + PTB was statistically comparable to isograft + suture, the current standard of care. Conclusion Previously reported advantages of PTB versus suture appear to be reduced when applied to ANA. The lack of Schwann cells and neurotrophic factors may be responsible. PTB may improve ANA performance to an extent, where they are equivalent to autograft. This may have important clinical implications when injuries preclude the use of autograft., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Photochemical tissue bonding uses visible light to create sutureless, watertight bonds between two apposed tissue surfaces stained with photoactive dye. When applied to nerve grafting, photochemical tissue bonding can result in superior outcomes compared with suture fixation. Our previous success has focused on immediate repair. It was the aim of this study to assess the efficacy of photochemical tissue bonding when performed following a clinically relevant delay., Methods: Forty male Lewis rats had 15-mm left sciatic nerve gaps repaired with reversed isografts immediately (n = 20) or after a 30-day delay (n = 20). Repairs were secured using either suture or photochemical tissue bonding. Rats were killed after 150 days. Outcomes were assessed using monthly Sciatic Function Index evaluation, muscle mass retention, and nerve histomorphometry. Statistical analysis was performed using analysis of variance and the post hoc Bonferroni test., Results: In both immediate and delayed groups, photochemical tissue bonding showed a trend toward greater recovery of Sciatic Function Index, but these results were not significant. The Sciatic Function Index was significantly greater when performed immediately. Significantly greater muscle mass retention occurred following photochemical tissue bonding in both immediate and delayed repairs. Values did not differ significantly between immediate and delayed groups. Histomorphometric recovery was greatest in the immediate photochemical tissue bonding group and poorest in the delayed suture group. Fiber diameter, axon diameter, myelin thickness, and G-ratio were not significantly different between immediate suture and delayed photochemical tissue bonding., Conclusions: Light-activated sealing of nerve grafts results in significantly better outcomes in comparison with conventional suture. The technique not only remains efficacious but may also help ameliorate the detrimental impacts of surgical delay.

Advances in photonics have stimulated significant progress in medicine, with many techniques now in routine clinical use. However, the finite depth of light penetration in tissue is a serious constraint to clinical utility. Here we show implantable light-delivery devices made of bio-derived or biocompatible, and biodegradable polymers. In contrast to conventional optical fibres, which must be removed from the body soon after use, the biodegradable and biocompatible waveguides may be used for long-term light delivery and need not be removed as they are gradually resorbed by the tissue. As proof of concept, we demonstrate this paradigm-shifting approach for photochemical tissue bonding (PTB). Using comb-shaped planar waveguides, we achieve a full thickness (>10 mm) wound closure of porcine skin, which represents ∼ 10-fold extension of the tissue area achieved with conventional PTB. The results point to a new direction in photomedicine for using light in deep tissues.

Background: Nerve repair using photochemically bonded human amnion nerve wraps can result in superior outcomes in comparison with standard suture. When applied to nerve grafts, efficacy has been limited by proteolytic degradation of bonded amnion during extended periods of recovery. Chemical cross-linking of amnion before bonding may improve wrap durability and efficacy., Methods: Three nerve wraps (amnion, cross-linked amnion, and cross-linked swine intestinal submucosa) and three fixation methods (suture, fibrin glue, and photochemical bonding) were investigated. One hundred ten Lewis rats had 15-mm left sciatic nerve gaps repaired with isografts. Nine groups (n = 10) had isografts secured by one of the aforementioned wrap/fixation combinations. Positive and negative control groups (n = 10) were repaired with graft and suture and no repair, respectively. Outcomes were assessed using sciatic function index, muscle mass retention, and histomorphometry. Statistical analysis was performed using analysis of variance and the post hoc Bonferroni test (p < 0.05)., Results: Cross-linking improved amnion durability. Photochemically bonded cross-linked amnion recovered the greatest sciatic function index, although this was not significant in comparison with graft and suture. Photochemically bonded cross-linked amnion recovered significantly greater muscle mass (67.3 ± 4.4 percent versus 60.0 ± 5.2 percent; p = 0.02), fiber diameter, axon diameter, and myelin thickness (6.87 ± 2.23 μm versus 5.47 ± 1.70 μm; 4.51 ± 1.83 μm versus 3.50 ± 1.44 μm; and 2.35 ± 0.64 μm versus 1.96 ± 0.47 μm, respectively) in comparison with graft and suture., Conclusion: Light-activated sealing of cross-linked human amnion results in superior outcomes when compared with conventional suture.

This study investigated a dual crosslinking paradigm, consisting of (a) photocrosslinking with Rose Bengal (RB) and green light followed by (b) chemical crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) to enhance collagen gel stiffness. In group 1, 50 μL collagen constructs of 2% (w/v) type I collagen containing 10 μM RB were allowed to gel spontaneously at 37 °C. In group 2, the spontaneous gels were exposed to green light (532 nm). In group 3, the photochemically crosslinked gels were subsequently treated with a 1-h exposure to 33 mM EDC/6 mM NHS. Samples (n = 18) were subjected to 0.08% (w/v) collagenase digestion, and the storage modulus of samples was measured by rheometry. Viability of encapsulated chondrocytes was measured by live/dead assay. Chondrocytes were ≥ 95% viable in all constructs at 10 days in vitro. Resistance to collagenase digestion increased as; spontaneous gels (2 h) < photochemical gels (3-4 h) < dual crosslinked gels (>24 h). The storage modulus of dual-crosslinked constructs was increased 5-fold over both photocrosslinked and spontaneous gels. As the dual crosslinking paradigm did not reduce encapsulated chondrocyte viability, these crosslinked collagen hydrogels could be a useful tool for the practical delivery of encapsulated chondrocytes to articular defects., (© 2014 Wiley Periodicals, Inc.)

Background and Objectives: We have developed a light-activated technology for rapidly sealing skin surgical wounds called photochemical tissue bonding (PTB). The goals of this study were to evaluate parameters influencing PTB in order to optimize its clinical efficacy and to determine whether PTB can be used to seal wounds in moderately to highly pigmented skin., Study Design/materials and Methods: Application of Rose Bengal (RB) followed by exposure to 532 nm was used to seal linear incisions (1.5 mm deep, 2 cm long) in lightly pigmented (Yorkshire) and darkly pigmented (Yucatan) swine skin. The force required to open the seal (the bonding strength) was measured by in situ tensiometry. Reflectance spectra, epidermal transmission spectra, and histology were used to characterize the skin. The relationships of RB concentration and fluence to bonding strength were established in Yorkshire skin. Surface temperature was measured during irradiations and cooling was used while sealing incisions in Yucatan skin. Monte Carlo simulations were carried out to estimate the effect of epidermal melanin on the power absorbed in the dermis at the incision interface., Results: The lowest fluence, 25 J/cm(2), delivered at an irradiance of 0.5 W/cm(2) substantially increased the bonding strength (∼ 10-fold) compared to controls in Yorkshire swine skin. Increasing the fluence to 100 J/cm(2) enhanced bonding strength by a further 1.5-fold. Application of 0.1% RB for 2 minutes produced the greatest bonding strength using 100 J/cm(2) and limited the penetration of RB to an ∼ 50 μm band on the dermal incision wall. Reflectance spectra indicated that Yorkshire skin had minimal melanin and that Yucatan skin was a good model for highly pigmented human skin. In Yucatan skin, the bonding strength increased 1.7-fold using 0.1% RB and 200 J/cm(2) at 1.5 W/cm(2) with cooling and epinephrine. Monte Carlo simulation indicated that absorption of 532 nm light by epidermal melanin in dark skin decreased the power absorbed along the incision in the dermis by a factor of 2.7., Conclusions: These results suggest that in lightly pigmented skin the PTB treatment time can be shortened without compromising the bonding strength. Sealing incisions using PTB in moderately and highly pigmented skin will require a careful balance of irradiance and cooling., (© 2014 Wiley Periodicals, Inc.)

Since the early 1900s, human amnion has been applied to a wide variety of clinical scenarios including burns, chronic ulcers, dural defects, intra-abdominal adhesions, peritoneal reconstruction, genital reconstruction, hip arthroplasty, tendon repair, nerve repair, microvascular reconstruction, corneal repair, intra-oral reconstruction and reconstruction of the nasal lining and tympanic membrane. Amnion epithelial and mesenchymal cells have been shown to contain a variety of regulatory mediators that result in the promotion of cellular proliferation, differentiation and epithelialisation and the inhibition of fibrosis, immune rejection, inflammation and bacterial invasion. The full repertoire of biological factors that these cells synthesise, store and release and the mechanisms by which these factors exert their beneficial effects are only now being fully appreciated. Although many commercially available biological and synthetic alternatives to amnion exist, ethical, religious, and financial constraints may limit the widespread utilisation of these products. Amnion is widely available, economical and is easy to manipulate, process and store. Although many clinical applications are of historical interest only, amnion offers an alternative source of multi-potent or pluripotent stem cells and therefore may yet have a great deal to offer the plastic surgery and regenerative medicine community. It is the purpose of this article to review the clinical applications of human amnion relevant to plastic surgery., (Copyright © 2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Long-wave UVA is the major component of terrestrial UV radiation and is also the predominant constituent of indoor sunlamps, both of which have been shown to increase cutaneous melanoma risk. Using a two-chamber model, we show that UVA-exposed target cells induce intercellular oxidative signaling to non-irradiated bystander cells. This UVA-mediated bystander stress is observed between all three cutaneous cell types (i.e., keratinocytes, melanocytes, and fibroblasts). Significantly, melanocytes appear to be more resistant to direct UVA effects compared with keratinocytes and fibroblasts, although melanocytes are also more susceptible to bystander oxidative signaling. The extensive intercellular flux of oxidative species has not been previously appreciated and could possibly contribute to the observed cancer risk associated with prolonged UVA exposure.

Photosensitized protein cross-linking has been recently developed to seal wounds and strengthen tissue. Although the photosensitizing dye, Rose Bengal (RB), is phototoxic to cultured cells, cytotoxicity does not accompany RB-photosensitized tissue repair in vivo. We investigated whether the environment surrounding cells in tissue or the high irradiances used for photo-cross-linking inhibited RB phototoxicity. Fibroblasts (FB) grown within collagen gels to mimic a tissue environment and monolayer cultured FB were treated with RB (0.01-1 mm) and the high 532 nm laser irradiances used in vivo for tissue repair (0.10-0.50 W cm(-2)). Monolayer FB were substantially more sensitive to RB photosensitization: the LD50 was >200-fold lower than that in collagen gels. Collagen gel protection was associated with increased Akt phosphorylation, a prosurvival pathway. RB phototoxicity in collagen gels was 25-fold greater at low (0.030 W cm(-2)) that at high (0.50 W cm(-2)) irradiances. Oxygen depletion at high irradiance only partially accounted for the irradiance dependence of phototoxicity as replacing air with nitrogen only increased the LD50 by four-fold in monolayers. These results indicate that the lack of RB phototoxicity during in vivo tissue repair results from upregulation of prosurvival pathways in tissue cells, oxygen depletion and irradiance-dependent RB photochemistry., (© 2013 The American Society of Photobiology.)

Background: Capsular contracture is the most common complication following the insertion of breast implants. Within a decade, half of patients will develop capsular contracture, leading to significant morbidity and need for reoperation. There is no preventative treatment available and the recurrence rate remains high. Photochemical tissue passivation is a novel tissue-stabilization technique that results in collagen cross-linking. It can rapidly link collagen fibers in situ, preserving normal tissue architecture. By using this therapy to passivate the collagenous tissues of the implant pocket, the authors hope to prevent the development of pathogenic collagen bundles and subsequent capsule contracture., Methods: Six-cubic centimeter tissue expanders were placed below the panniculus carnosus muscle along the dorsum of New Zealand white rabbits. Fibrin glue was instilled into each implant pocket to induce contracture. Treated pockets received photochemical tissue passivation by coating them with a photosensitizing dye and exposing the area to a 532-nm light. After 8 weeks, capsule tissue was harvested for histologic evaluation., Results: Implant capsule thickness is the number one prognostic factor for contracture development. The authors demonstrated a 52 percent decrease in capsule thickness in the passivated group compared with controls. Photochemical tissue passivation resulted in fewer fibrohistiocytic cells and macrophages and in reduced synovial metaplasia and smooth muscle actin deposition., Conclusions: Photochemical tissue passivation significantly decreased both capsule thickness and smooth muscle actin deposition. It is a promising technique for preventing capsular contracture that can be performed at the time of initial surgery without a significant increase in procedure time.

Purpose: Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application., Methods: Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment., Results: RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability., Conclusions: RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.

Background and Objective: Suture repair of Achilles tendon rupture can cause infection, inflammation and scarring, while prolonged immobilization promotes adhesions to surrounding tissues and joint stiffness. Early mobilization can reduce complications provided the repair is strong enough to resist re-rupture. We have developed a biocompatible, photoactivated tendon wrap from electrospun silk (ES) to provide additional strength to the repair that could permit early mobilization, and act as a barrier to adhesion formation., Study Design/material and Methods: ES nanofiber mats were prepared by electrospinning. New Zealand white rabbits underwent surgical transection of the Achilles tendon and repair by: (a) SR: standard Kessler suture + epitendinous suture (5-0 vicryl). (b) ES/PTB: a single stay suture and a section of ES mat, stained with 0.1% Rose Bengal (RB), wrapped around the tendon and bonded with 532 nm light (0.3 W/cm(2) , 125 J/cm(2) ). (c) SR + ES/PTB: a combination of (a) and (b). Gross appearance, extent of adhesion formation and biomechanical properties of the repaired tendon were evaluated at Days 7, 14, or 28 post-operatively (n = 8 per group at each time point)., Results: Ultimate stress (US) and Young's modulus (E) in the SR group were not significantly different from the ES/PTB group at Days 7 (US, P = 0.85; E, P = 1), 14 (US, P = 0.054; E, P = 1), and 28 (US, P = 0.198; E, P = 0.12) post-operatively. Adhesions were considerably greater in the SR group compared to the ES/PTB group at Days 7 (P = 0.002), 14 (P < 0.0001), and 28 (P < 0.0001). The combination approach of SR + ES/PTB gave the best outcomes in terms of E at 7 (P < 0.016) and 14 days (P < 0.016) and reduced adhesions compared to SR at 7 (P < 0.0001) and 14 days (P < 0.0001), the latter suggesting a barrier function for the photobonded ES wrap., Conclusion: Photochemical sealing of a ES mat around the tendon repair site provides considerable benefit in Achilles tendon repair. Lasers Surg. Med. 44: 645-652, 2012. © 2012 Wiley Periodicals, Inc., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Apposition of wound edges by sutures provides a temporary scaffold and tension support for healing. We have developed a novel tissue-sealing technology, photoactivated tissue bonding (PTB), which immediately crosslinks proteins between tissue planes, thereby sealing on a molecular scale., Objectives: To determine the effectiveness of PTB for superficial closure of skin excisions and to compare the results with standard epidermal suturing., Methods: A split-lesion, paired comparison study of 31 skin excisions was performed. Following deep closure with absorbable sutures, one-half of each wound was superficially closed with nonabsorbable nylon sutures while the other half was stained with Rose Bengal dye and treated with green light. Overall appearance and scar characteristics were rated at 2weeks and 6months in a blinded manner by three dermatologists viewing photographs, by two onsite physicians and by patients., Results: At 2weeks, neither sutured nor PTB-treated segments showed dehiscence; however, PTB-sealed segments showed less erythema than sutured segments as determined by photographic (P=0·001) and onsite evaluations (P=0·005). Overall appearance after PTB was judged better than after sutures (P=0·002). At 6months, scars produced by PTB were deemed superior to scars resulting from sutures in terms of appearance (P<0·001), width (P=0·002) and healing (P=0·003). Patients were more satisfied with the appearance of the PTB-sealed wound half after 2weeks and 6months (P=0·013 and P=0·003, respectively)., Conclusions: A novel molecular suturing technique produces effective wound sealing and less scarring than closure with nylon interrupted epidermal sutures. Comparisons with better suturing techniques are warranted., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

Background and Objectives: Closing lacerations in thin eyelid and periorbital skin is time consuming and requires high skill for optimal results. In this study we evaluate the outcomes after single layer closure of wounds in thin skin with a sutureless, light-activated photochemical technique called PTB., Study Design/materials and Methods: Dorsal skin of the SKH-1 hairless mouse was used as a model for eyelid skin. Incisions (1.2 cm) were treated with 0.1% Rose Bengal dye followed by exposure to 532 nm radiation (25, 50, or 100 J/cm(2); 0.25 W/cm(2)) for PTB. Other incisions were sutured (five 10-0 monofilament), exposed only to 532 nm (100 J/cm(2)), or not treated. Outcomes were immediate seal strength (pressure causing leakage through incision of saline infused under wound), skin strength at 1, 3, and 7 days (measured by tensiometry), inflammatory infiltrate at 1, 3, and 7 days (histological assessment), and procedure time., Results: The immediate seal strength, as measured by leak pressure, was equivalent for all PTB fluences and for sutures (27-32 mmHg); these pressures were significantly greater than for the controls (untreated incisions or laser only treatment; P < 0.001). The ultimate strength of PTB-sealed incisions was greater than the controls at day 1 (P < 0.05) and day 3 (P < 0.025) and all groups were equivalent at day 7. Sutures produced greater inflammatory infiltrate at day 1 than observed in other groups (P = 0.019). The average procedure time for sutured closure (311 seconds) was longer than for the PTB group treated with 25 J/cm(2) (160 seconds) but shorter than the group treated with 100 J/cm(2) (460 seconds)., Conclusion: PTB produces an immediate seal of incisions in thin, delicate skin that heals well, is more rapid than suturing, does not require painful suture removal and is easy to apply., (Copyright © 2011 Wiley Periodicals, Inc.)

Purpose: Suturing amniotic membrane to cornea during surgery is time consuming, and sutures may further damage the eye. The authors introduce a novel sutureless, light-activated technique that securely attaches amnion to cornea through protein-protein crosslinks., Methods: Cryopreserved human amniotic membrane, stained with Rose Bengal (RB), was placed over a full-thickness wound in deepithelialized rabbit cornea and was treated with green laser. The intraocular pressure that broke the seal (IOP(L)) was measured, and adhesion was measured with a peel test. The influences on bonding strength of fluence, irradiance, RB concentration, and amnion surface bonded were measured. Epithelial cell migration on treated amnion and keratocyte viability after bonding were also measured. The involvement in the bonding mechanism of oxygen, singlet oxygen, and association of RB with stromal collagen was investigated., Results: Sealing amniotic membrane over cornea using 0.1% RB and 150 J/cm(2) at 532 nm produced an IOP(L) of 261 ± 77 mm Hg ex vivo and 448 mm ± 212 mm Hg in vivo. The ex vivo IOP(L) increased with increasing fluence (50-150 J/cm(2)). Equivalent IOP(L) was produced for bonding basement membrane or stromal amnion surfaces. The bonding treatment was not toxic to keratocytes but slightly reduced the migration of corneal epithelial cells on amnion ex vivo. Mechanism studies indicated that RB forms two complexes with amnion stromal collagen, that bonding requires oxygen, and that singlet oxygen mediates protein crosslinking., Conclusions: A rapid, light-activated technique produces strong, immediate bonding between amnion and cornea and merits further evaluation for ocular surface surgeries.

Background and Objective: Amniotic membrane transplantation (AMT) sealed with sutures has been routinely used to treat ocular surface defects (OSD). However, the sutures used to secure the graft on the cornea cause additional injury, infection, and scarring. A new light-activated technique, called photochemical tissue bonding (PTB), has been developed for securing amniotic membrane (AM) over the corneal surface. The purpose of this study was to compare PTB versus traditional sutures in AMT for repair of OSD., Materials and Methods: An OSD was created in the left eye of 40 rabbits. The eyes were randomized into two repair groups: AMT using sutures and AMT using PTB with 0.1% Rose Bengal (RB) and 532 nm laser at 0.4 W/cm(2) for 200 seconds. Eyes were examined for re-epithelialization, inflammation, neovascularization, and scarring histologically and biochemically on postoperative days 1, 3, 14, and 28., Results: PTB strongly bonded AM over corneal defects. Corneal re-epithelialization did not differ significantly between the suture and PTB groups. Histology, immunohistology and Western blotting revealed that the numbers of inflammatory cells and the level of tumor necrosis factor-alpha in the PTB group were dramatically lower than those in the suture group on postoperative day 3. Many fewer neo-vessels were present in the PTB group (2.91 ± 1.00) compared to the suture group (4.33 ± 1.15) at day 28 (P < 0.05). The collagen fibers in the PTB group were well organized and orientated as assessed by second harmonic generation microscopy, suggesting that PTB treatment led to less corneal scarring., Conclusions: PTB is a superior method for securing AM over OSD with improved wound healing compared to sutures., (Copyright © 2011 Wiley-Liss, Inc.)

Background and Objective: To determine the feasibility of attaching human amniotic membrane (HAM), pre-cultured with limbal stem cells (LSCs), to cornea using a novel, light-activated tissue bonding method., Materials and Methods: LSCs were isolated from rabbit eyes, and then cultured on de-epithelialized HAM to create grafts (HAM/LSC). These were then transplanted onto rabbit eyes with surgically created limbal stem cell deficiency (LSCD). The grafts were secured either by sutures or by a light-activated method called photochemical tissue bonding (PTB). Outcomes included corneal opacity, inflammation, neovascularization, and collagen alignment., Results: The isolated and cultured cells were verified to be LSCs based on their K19+/intergrin β1+/P63+/K3 profile. Securing the HAM/LSC graft with PTB provided better outcomes. At 28 days post-surgery, the corneal opacity scores were significantly lower after securing the graft with PTB compared with suture attachment (0.8 ± 0.5 vs. 1.8 ± 0.5, P < 0.01). Similarly, neovascularization scores were lower after PTB (0.8 ± 0.5 vs. 1.5 ± 0.6, P < 0.01). Quantification of MPO and CD31 levels from immunofluorecent staining indicated that PTB stimulated less neutrophil infiltration (5.3 ± 2.2 vs. 13.3 ± 3.1, P < 0.01) and less new blood vessels formation (2.0 ± 0.8 vs. 6.3 ± 1.3, P < 0.01) at the wound site. The collagen alignment in PTB-treated corneas, as shown by immunofluorescence and second harmonic generation image, was better organized in the PTB-treated group than in the suture group., Conclusion: Bonding LSC grafts with PTB produced improved outcomes compared to suture attachment. This light-activated method is a promising modality for treating patients with LSCD., (Copyright © 2011 Wiley-Liss, Inc.)

Objectives: To bond vocal fold flaps using a sutureless, nonthermal laser-assisted method combining visible light and photosensitizing dyes to produce collagen crosslinks., Study Design: In the ex vivo study, epithelial incisions were created in sheep vocal folds. The concentration of the Rose Bengal, and the laser fluence were varied and studied. The in vivo canine study evaluated the thickness of the basement membrane zone, density and distribution of collagen, elastin, and fibroblasts at time zero, 2 weeks, and 8 weeks., Methods: Ex Vivo: Rose Bengal at concentrations between 0.5% and 1.0% was applied to the free margins of the incision and irradiated with an Nd:YAG laser at fluences varying from 150 to 600 J/cm2 . The bonding was considered positive when the incision could withstand air at 2 pounds per square inch (psi) from a distance of 3 cm. In Vivo: 0.75% Rose Bengal was applied to the microflap edges and irradiated with an Nd:YAG laser for 140 seconds (100 J/cm2). The control side was not irradiated., Results: Ex Vivo: Bonding was achieved with a minimum of 0.75% Rose Bengal and 300 J/cm2 . In Vivo: There was no difference in the amount of subepithelial reaction between the experimental and control sides at 8 weeks., Conclusions: Photochemical tissue bonding is effective at sealing vocal fold incisions and did not create long-term scarring of the vocal folds. Use of this technique may allow for more predictable healing after microflap resection and may reduce the need for postoperative voice rest., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.

Dyes that bind to DNA, such as Hoechst 33342, are commonly used to visualize chromatin in live cells by fluorescence microscopy. A caveat is that the probes themselves should not perturb cellular responses and under normal conditions the dyes are generally non-toxic. However, researchers are increasingly using computerized time-lapse microscopy (CTLM), where cells stained with fluorescent dyes are often imaged frequently over a period of several days, to follow cellular responses in real time. Little is currently known about possible toxicity of fluorescent DNA dyes under CTLM conditions. In this study we demonstrate that the common live-cell DNA stain Hoechst 33342 can cause apoptosis under CTLM conditions. Although toxicity is evident at long times in the absence of imaging at high dye concentrations, phototoxicity from repeated excitation of the dye in the imaging process is dominant. We show that phototoxicity is a function of the product of light fluence and dye concentration, irrespective of irradiance, frequency and total number of scans. Thus, phototoxicity can be prevented by a combination of dye concentration and imaging procedure that is below this threshold. These quantitative data can be used as a guide to others performing time-lapse microscopy studies with this common live-cell DNA stain and serves as a caution for researchers when using other fluorescent stains under CTLM conditions.

Motor unit number estimation (MUNE) allows for quantitative assessment of functional motor units in a nerve. Several techniques have been applied to human studies. Although MUNE has been performed in animals to study neurological disorders, reproducibility has not been addressed. We analyzed the test-retest reproducibility of an incremental MUNE technique in rabbits and performed histological correlation. A peroneal MUNE was performed in 9 rabbits on two occasions separated by 30 days. MUNE was then performed on 18 rabbits prior to euthanize. A count of total fibers and a second count of large myelinated fibers were performed on nerve cross-sections. Test-retest reproducibility revealed an intraclass correlation coefficient (ICC) of 0.75. The average test-retest relative difference was 26.6%. Comparison of MUNE and histomorphometrical counts revealed a correlation coefficient (r) of 0.21 (total fiber counts) and 0.27 (large fibers). Although incremental MUNE has a high degree of reproducibility in rabbits, there is poor correlation with histological fiber counts.

Background and Objective: We have developed a light-activated method called photochemical tissue bonding (PTB) for closing wounds using green light and a photosensitizing dye (Rose Bengal-RB) to initiate photochemical crosslinking of wound surface proteins. These studies were designed to determine whether RB causes phototoxicity during closure of skin incisions with PTB., Study Design/materials and Methods: RB phototoxicity was evaluated after sealing incisions in porcine skin ex vivo and rabbit skin in vivo using PTB (1 mM RB, 100 J/cm(2), 532 nm, 0.3 or 0.5 W/cm(2).) Dead cells were identified by pyknotic nuclei and eosinophilic cytoplasm on H&E-stained sections. The influence on RB phototoxicity of penetration of RB into the wound wall (by confocal microscopy), RB concentration in the tissue (by extraction), and fluence of 532 nm reaching depths in skin (calculated from skin optical properties) were investigated., Results: No significant differences were found in the percent dead cells in PTB-treated and control incisions in porcine skin at 24 hours or in rabbit skin at 2 hours and 3 and 7 days after surgery. RB was retained in a approximately 100 microm wide band next to the wound wall. The mean RB concentration within this band was 0.42+/-0.03 mM. Monte Carlo modeling of light distribution indicated that the fluence rate decreased from the subsurface peak to 0.5 W/cm(2) in the mid-dermis (approximately 350 microm.) In vitro RB phototoxicity to dermal fibroblasts yielded an LD(50) of 0.50+/-0.09 J/cm(2) when the cells contained 0.46 mM RB., Conclusions: PTB does not cause phototoxicity when used to repair skin wounds even though the RB concentration and 532 nm fluence in the mid-dermis during PTB are much greater than the LD(50) for RB phototoxicity in vitro. These results indicate that phototoxicity is not a concern when using PTB for tissue repair.

This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naïve recipient cells. Kinetic studies revealed that it required up to 1h to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least 1h of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12-24h to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent gamma-ray exposures.

Cells subjected to various forms of stress have been shown to induce bystander responses in nontargeted cells, thus extending the stress response to a larger population. However, the mechanism(s) of bystander responses remains to be clearly identified, particularly for photodynamic stress. Oxidative stress and cell viability were studied on the spatial and temporal levels after photodynamic targeting of a subpopulation of EMT6 murine mammary cancer cells in a multiwell plate by computerized time-lapse fluorescence microscopy. In the targeted population a dose-dependent loss of cell viability was observed in accordance with increased oxidative stress. This was accompanied by increased oxidative stress in bystander populations but on different time scales, reaching a maximum more rapidly in targeted cells. Treatment with extracellular catalase, or the NADPH oxidase inhibitor diphenyleneiodinium, decreased production of reactive oxygen species (ROS) in both populations. These effects are ascribed to photodynamic activation of NADPH-oxidase in the targeted cells, resulting in a rapid burst of ROS formation with hydrogen peroxide acting as the signaling molecule responsible for initiation of these photodynamic bystander responses. The consequences of increased oxidative stress in bystander cells should be considered in the overall framework of photodynamic stress.

Background: Photochemical tissue bonding is a developing light-activated technique that facilitates watertight sealing between tissue surfaces. Previous work has shown that sealing with photochemical tissue bonding can improve regeneration following primary nerve repair. The authors evaluated sealing of nerve stumps with photochemical tissue bonding within customized human amnion conduits. The authors hypothesized that light-activated integration could enhance regeneration across the nerve gap., Methods: Photochemical crosslinked amnion conduits were placed across 1-cm sciatic nerve gaps in Sprague-Dawley rats and either secured with sutures or sealed using photochemical tissue bonding. Reversed autologous nerve grafts were used in the control group. Functional recovery was measured by walking track analysis; histology and histomorphometry of nerves and gastrocnemius muscles were evaluated., Results: Regeneration within the photochemical tissue bonding-sealed amnion conduit was significantly better than that observed in the amnion conduit secured with sutures and did not differ significantly from that in the autologous nerve graft., Conclusions: Photochemical crosslinked amnion appears suitable as a nerve conduit. Sealing of compatible conduits with photochemical tissue bonding may have the potential to maximize regeneration.

The bystander effect, whereby cells that are not traversed by ionizing radiation exhibit various responses when in proximity to irradiated cells, is well documented in the field of radiation biology, Here we demonstrate that considerable bystander responses are also observed after photodynamic stress using the membrane-localizing dye deuteroporphyrin (DP). Using cells of a WTK1 human lymphoblastoid cell line in suspension and a transwell insert system that precludes contact between targeted and bystander cells, we have shown that the bystander signaling is mediated by diffusing species. The extranuclear localization of the photosensitizer used suggests that primary DNA damage is not the trigger for initiating these bystander responses, which include elevated oxidative stress, DNA damage (micronucleus formation), mutagenesis and decreased clonogenic survival. In addition, oxidative stress in the bystander population was reduced by the presence of the membrane antioxidant vitamin E in the targeted cells, suggesting that lipid peroxidation may play a key role in mediating these bystander effects. The fluence responses for these bystander effects are non-linear, with larger effects seen at lower fluences and toxicity to the target cell population. Hence, when considering outcomes of photodynamic action in cells and tissue, bystander effects may be significant, especially at sublethal fluences.