Person:

Randolph, Mark

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: Injuries to the human native cartilage tissue are particularly problematic because cartilage has little to no ability to heal or regenerate itself. Employing a tissue engineering strategy that combines suitable cell sources and biomimetic hydrogels could be a promising alternative to achieve cartilage regeneration. However, the weak mechanical properties may be the major drawback to use fully degradable hydrogels. Besides, most of the fully degradable hydrogels degrade too fast to permit enough extracellular matrix (ECM) production for neocartilage formation. In this study, we demonstrated the feasibility of neocartilage regeneration using swine articular chondrocytes photoencapsualted into poly (ethylene glycol) dimethacrylate (PEGDM) copolymer hydrogels composed of different degradation profiles: degradable (PEG-LA-DM) and nondegradable (PEGDM) macromers in molar ratios of 50/50, 60/40, 70/30, 80/20, and 90/10. Methods: Articular chondrocytes were isolated enzymatically from juvenile Yorkshire swine cartilage. 6 × 107 cells cells were added to each milliliter of macromer/photoinitiator (I2959) solution. Nonpolymerized gel containing the cells (100 μL) was placed in cylindrical molds (4.5 mm diameter × 6.5 mm in height). The macromer/photoinitiator/chondrocyte solutions were polymerized using ultraviolet (365 nm) light at 10 mW/cm2 for 10 mins. Also, an articular cartilaginous ring model was used to examine the capacity of the engineered cartilage to integrate with native cartilage. Samples in the pilot study were collected at 6 weeks. Samples in the long-term experimental groups (60/40 and 70/30) were implanted into nude mice subcutaneously and harvested at 6, 12 and 18 weeks. Additionally, cylindrical constructs that were not implanted used as time zero controls. All of the harvested specimens were examined grossly and analyzed histologically and biochemically. Results: Histologically, the neocartilage formed in the photochemically crosslinked gels resembled native articular cartilage with chondrocytes in lacunae and surrounded by new ECM. Increases in total DNA, glycosaminoglycan, and hydroxyproline were observed over the time periods studied. The neocartilage integrated with existing native cartilage. Conclusions: Articular cartilage generation was achieved using swine articular chondrocytes photoencapsulated in copolymer PEGDM hydrogels, and the neocartilage tissue had the ability to integrate with existing adjacent native cartilage.

Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.

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/cm2 on porcine veins. Tensiometry performed to assess vessel elasticity/stiffness showed increased stiffness with increasing fluence until plateauing at 90 J/cm2 (median, interquartile range [IQR]). At 90 J/cm2, 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 mm2 (IQR, 0.97; P≤0.001) in untreated and PTP‐treated grafts, respectively. Medial area was 9.2 (IQR, 3.2) and 4.7 mm2 (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.

Objective: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice. Design: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed. Results: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks. Conclusions: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

For patients with extensive burns or donor site scarring, the limited availability of autologous and the inevitable rejection of allogeneic skin drive the need for new alternatives. Existing engineered biologic and synthetic skin analogs serve as temporary coverage until sufficient autologous skin is available. Here we report successful engraftment of a self-assembled bilayered skin construct derived from autologous skin punch biopsies in a porcine model. Dermal fibroblasts were stimulated to produce an extracellular matrix and were then seeded with epidermal progenitor cells to generate an epidermis. Autologous constructs were grafted onto partial- and full-thickness wounds. By gross examination and histology, skin construct vascularization and healing were comparable to autologous skin grafts and were superior to an autologous bilayered living cellular construct fabricated with fibroblasts cast in bovine collagen. This is the first demonstration of spontaneous vascularization and permanent engraftment of a self-assembled bilayered bioengineered skin that could supplement existing methods of reconstruction.