Histologic Status of Squamous Cell Carcinoma In Situ After Diagnostic Biopsy in Immunocompetent and Immunosuppressed Patients.

BACKGROUND: The histologic status of squamous cell carcinoma in situ (SCC-IS) after diagnostic biopsy has not been well described or compared between immunocompetent and immunosuppressed patients. Expression of immunohistochemical (IHC) markers of aggressive SCC has not been compared between SCC-IS that clears or becomes invasive after biopsy. OBJECTIVE: To determine the histologic status of SCC-IS after diagnostic biopsy in these populations. METHODS: Retrospective analysis of 129 patients with SCC-IS treated with excision and 55 patients treated with Mohs surgery. Histologic features of SCC in excised tissue after biopsy were recorded. Known SCC markers were evaluated using IHC. RESULTS: Invasive SCC was found in 3% to 16% of residual SCC-IS depending on surgical treatment modality. The history of skin cancer increased the odds of having invasive SCC in SCC-IS excisions (odds ratio 7.1, p < .05). Forty-seven percent of SCC-IS in immunosuppressed patients cleared after diagnostic biopsy compared with 70% in immunocompetent patients (p < .05). Inflammatory infiltrate and molecular markers of aggressive SCCs (Ki-67, matrix metalloproteinase [MMP]-9, MMP-7, transforming growth factor-beta (TGFß)-RI, TGFß-RII, and Sox-2) were not predictive of residual or invasive SCC at the time of treatment. CONCLUSION: Up to 16% of SCC-IS showed invasive SCC at the time of surgical treatment. Immunosuppressed patients are more likely to have residual disease after biopsy. IHC markers of invasive SCC may not predict invasion.

Regenerative Medicine and Stem Cells in Dermatology.

BACKGROUND: Clinically relevant regenerative medicine is still in its early stages of development. Difficulties in regenerating large-scale and complex structures, the lack of safety data, and the paucity of clinical trials have slowed the process of technological advance. OBJECTIVE: To familiarize the clinician with techniques available in the laboratory and experimental approaches being tested clinically. In addition, a layout is discussed for how dermatologists can lead the way in bringing regenerative medicine to clinical reality. METHODS: This article reviews the relevant literature on regenerative medicine for dermatological applications and discusses findings and techniques in a clinically relevant context. RESULTS: Multiple cell-free and cell-based approaches for regenerating dermatologic tissues have been reported in the basic science and clinical literature. These are reviewed in the order of complexity. CONCLUSION: Incremental steps are needed to apply the principles of regenerative medicine to simple medical problems first. Such a stepwise approach would commence, for example, with creation of single-function tissues that could fill soft-tissue defects and proceed to the development of fully functional skin grafts. Likewise, cell-free approaches can build the foundation for the more technically demanding cell-based strategies that are likely necessary for achieving the ultimate goal of regenerative dermatology.

Nonmelanoma Skin Cancer in Patients Older Than Age 85 Years Presenting for Mohs Surgery: A Prospective, Multicenter Cohort Study.

Importance: It has been suggested that Mohs surgery for skin cancer among individuals with limited life expectancy may be associated with needless risk and discomfort, along with increased health care costs. Objective: To investigate patient- and tumor-specific indications considered by clinicians for treatment of nonmelanoma skin cancer in older individuals. Design, Setting, and Participants: This multicenter, prospective cohort study was conducted using data from US private practice and academic centers. Included patients were those older than age 85 years presenting for skin cancer surgery and referred for Mohs surgery, with reference groups of those younger than age 85 years receiving Mohs surgery and those older than age 85 years not receiving Mohs surgery. Data were analyzed from November 2018 through January 2019. Exposures: Mohs surgery for nonmelanoma skin cancer. Main Outcomes and Measures: Reason for treatment selection. Results: Among 1181 patients older than age 85 years referred for Mohs surgery (724 [61.9%] men among 1169 patients with sex data; 681 individuals aged >85 to 88 years [57.9%] among 1176 patients with age data) treated at 22 sites, 1078 patients (91.3%) were treated by Mohs surgery, and 103 patients (8.7%) received alternate treatment. Patients receiving Mohs surgery were more likely to have tumors on the face (738 patients [68.5%] vs 26 patients [25.2%]; P < .001) and nearly 4-fold more likely to have high functional status (614 patients [57.0%] vs 16 patients [15.5%]; P < .001). Of 15 distinct reasons provided by surgeons for opting to proceed with Mohs surgery, the most common were patient desire for treatment with a high cure rate (712 patients [66.0%]), good or excellent patient functional status for age (614 patients [57.0%]), and high risk associated with the tumor based on histology (433 patients [40.2%]). Conclusions and Relevance: This study found that older patients who received Mohs surgery often had high functional status, high-risk tumors, and tumors located on the face. These findings suggest that timely surgical treatment may be appropriate in older patients given that their tumors may be aggressive, painful, disfiguring, and anxiety provoking.

Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study.

BACKGROUND: A common approach for tissue regeneration is cell delivery, for example by direct transplantation of stem or progenitor cells. An alternative, by recruitment of endogenous cells, needs experimental evidence. We tested the hypothesis that the articular surface of the synovial joint can regenerate with a biological cue spatially embedded in an anatomically correct bioscaffold. METHODS: In this proof of concept study, the surface morphology of a rabbit proximal humeral joint was captured with laser scanning and reconstructed by computer-aided design. We fabricated an anatomically correct bioscaffold using a composite of poly-epsilon-caprolactone and hydroxyapatite. The entire articular surface of unilateral proximal humeral condyles of skeletally mature rabbits was surgically excised and replaced with bioscaffolds spatially infused with transforming growth factor beta3 (TGFbeta3)-adsorbed or TGFbeta3-free collagen hydrogel. Locomotion and weightbearing were assessed 1-2, 3-4, and 5-8 weeks after surgery. At 4 months, regenerated cartilage samples were retrieved from in vivo and assessed for surface fissure, thickness, density, chondrocyte numbers, collagen type II and aggrecan, and mechanical properties. FINDINGS: Ten rabbits received TGFbeta3-infused bioscaffolds, ten received TGFbeta3-free bioscaffolds, and three rabbits underwent humeral-head excision without bioscaffold replacement. All animals in the TGFbeta3-delivery group fully resumed weightbearing and locomotion 3-4 weeks after surgery, more consistently than those in the TGFbeta3-free group. Defect-only rabbits limped at all times. 4 months after surgery, TGFbeta3-infused bioscaffolds were fully covered with hyaline cartilage in the articular surface. TGFbeta3-free bioscaffolds had only isolated cartilage formation, and no cartilage formation occurred in defect-only rabbits. TGFbeta3 delivery yielded uniformly distributed chondrocytes in a matrix with collagen type II and aggrecan and had significantly greater thickness (p=0.044) and density (p<0.0001) than did cartilage formed without TGFbeta3. Compressive and shear properties of TGFbeta3-mediated articular cartilage did not differ from those of native articular cartilage, and were significantly greater than those of cartilage formed without TGFbeta3. Regenerated cartilage was avascular and integrated with regenerated subchondral bone that had well defined blood vessels. TGFbeta3 delivery recruited roughly 130% more cells in the regenerated articular cartilage than did spontaneous cell migration without TGFbeta3. INTERPRETATION: Our findings suggest that the entire articular surface of the synovial joint can regenerate without cell transplantation. Regeneration of complex tissues is probable by homing of endogenous cells, as exemplified by stratified avascular cartilage and vascularised bone. Whether cell homing acts as an adjunctive or alternative approach of cell delivery for regeneration of tissues with different organisational complexity warrants further investigation. FUNDING: New York State Stem Cell Science; US National Institutes of Health.

Bioengineering strategies to generate vascularized soft tissue grafts with sustained shape.

Tissue engineering offers the possibility for soft tissue reconstruction and augmentation without autologous grafting or conventional synthetic materials. Two critical challenges have been addressed in a number of recent studies: a biology challenge of angiogenesis and an engineering challenge of shape maintenance. These two challenges are inter-related and are effectively addressed by integrated bioengineering strategies. Recently, several integrated bioengineering strategies have been applied to improve bioengineered adipose tissue grafts, including internalized microchannels, delivery of angiogenic growth factors, tailored biomaterials and transplantation of precursor cells with continuing differentiation potential. Bioengineered soft tissue grafts are only clinically meaningful if they are vascularized, maintain shape and dimensions, and remodel with the host. Ongoing studies have begun to demonstrate the feasibility towards an ultimate goal to generate vascularized soft tissue grafts that maintain anatomically desirable shape and dimensions.

Porous implants as drug delivery vehicles to augment host tissue integration.

The common premise of synthetic implants in the restoration of diseased tissues and organs is to use inert and solid materials. Here, a porous titanium implant was fabricated for the delivery of microencapsulated bioactive cues. Control-released transforming growth factor-beta1 (TGF-beta1) promoted the proliferation and migration of human mesenchymal stem cells into porous implants in vitro. At 4 wk of implantation in the rabbit humerus, control-released TGF-beta1 from porous implants significantly increased bone-to-implant contact (BIC) by 96% and bone ingrowth by 50% over placebos. Control-released 100 ng TGF-beta1 induced equivalent BIC and bone ingrowth to adsorbed 1 microg TGF-beta1, suggesting that controlled release is effective at 10-fold less drug dose than adsorption. Histomorphometry, scanning electron microscopy, and microcomputed tomography showed that control-released TGF-beta1 enhanced bone ingrowth in the implant's pores and surface. These findings suggest that solid prostheses can be transformed into porous implants to serve as drug delivery carriers, from which control-released bioactive cues augment host tissue integration.

Autologous stem cell regeneration in craniosynostosis.

Craniosynostosis occurs in one of 2500 live human births and may manifest as craniofacial disfiguration, seizure, and blindness. Craniotomy is performed to reshape skull bones and resect synostosed cranial sutures. We demonstrate for the first time that autologous mesenchymal stem cells (MSCs) and controlled-released TGFbeta3 reduced surgical trauma to localized osteotomy and minimized osteogenesis in a rat craniosynostosis model. Approximately 0.5 mL tibial marrow content was aspirated to isolate mononucleated and adherent cells that were characterized as MSCs. Upon resecting the synostosed suture, autologous MSCs in collagen carriers with microencapsulated TGFbeta3 (1 ng/mL) generated cranial suture analogs characterized as bone-soft tissue-bone interface by quantitative histomorphometric and microCT analyses. Thus, surgical trauma in craniosynostosis can be minimized by a biologically viable implant. We speculate that proportionally larger amounts of human marrow aspirates participate in the healing of craniosynostosis defects in patients. The engineered soft tissue-bone interface may have implications in the repair of tendons, ligaments, periosteum and periodontal ligament.

Matrices and scaffolds for drug delivery in dental, oral and craniofacial tissue engineering.

Current treatments for diseases and trauma of dental, oral and craniofacial (DOC) structures rely on durable materials such as amalgam and synthetic materials, or autologous tissue grafts. A paradigm shift has taken place to utilize tissue engineering and drug delivery approaches towards the regeneration of these structures. Several prototypes of DOC structures have been regenerated such as temporomandibular joint (TMJ) condyle, cranial sutures, tooth structures and periodontium components. However, many challenges remain when taking in consideration the high demand for esthetics of DOC structures, the complex environment and yet minimal scar formation in the oral cavity, and the need for accommodating multiple tissue phenotypes. This review highlights recent advances in the regeneration of DOC structures, including the tooth, periodontium, TMJ, cranial sutures and implant dentistry, with specific emphasis on controlled release of signaling cues for stem cells, biomaterial matrices and scaffolds, and integrated tissue engineering approaches.

Sustained release of TGFbeta3 from PLGA microspheres and its effect on early osteogenic differentiation of human mesenchymal stem cells.

Despite the widespread role of transforming growth factor-beta3 (TGFbeta3) in wound healing and tissue regeneration, its long-term controlled release has not been demonstrated. Here, we report microencapsulation of TGFbeta3 in poly-d-l-lactic-co-glycolic acid (PLGA) microspheres and determine its bioactivity. The release profiles of PLGA-encapsulated TGFbeta3 with 50:50 and 75:25 PLA:PGA ratios differed throughout the experimental period. To compare sterilization modalities of microspheres, bFGF was encapsulated in 50:50 PLGA microspheres and subjected to ethylene oxide (EO) gas, radio-frequency glow discharge (RFGD), or ultraviolet (UV) light. The release of bFGF was significantly attenuated by UV light, but not significantly altered by either EO or RFGD. To verify its bioactivity, TGFbeta3 (1.35 ng/mL) was control-released to the culture of human mesenchymal stem cells (hMSC) under induced osteogenic differentiation. Alkaline phosphatase staining intensity was markedly reduced 1 week after exposing hMSC-derived osteogenic cells to TGFbeta3. This was confirmed by lower alkaline phosphatase activity (2.25 +/- 0.57 mU/mL/ng DNA) than controls (TGFbeta3- free) at 5.8 +/- 0.9 mU/mL/ng DNA (p < 0.05). Control-released TGFbeta3 bioactivity was further confirmed by lack of significant differences in alkaline phosphatase upon direct addition of 1.35 ng/mL TGFbeta3 to cell culture (p > 0.05). These findings provide baseline data for potential uses of microencapsulated TGFbeta3 in wound healing and tissue-engineering applications.

CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model.

Fibroblasts are ubiquitous cells that demonstrate remarkable diversity. However, their origin and pathways of differentiation remain poorly defined. Here, we show that connective tissue growth factor (CTGF; also known as CCN2) is sufficient to induce human bone marrow mesenchymal stem/stromal cells (MSCs) to differentiate into fibroblasts. CTGF-stimulated MSCs lost their surface mesenchymal epitopes, expressed broad fibroblastic hallmarks, and increasingly synthesized collagen type I and tenacin-C. After fibroblastic commitment, the ability of MSCs to differentiate into nonfibroblastic lineages - including osteoblasts, chondrocytes, and adipocytes - was diminished. To address inherent heterogeneity in MSC culture, we established 18 single MSC-derived clones by limiting dilution. CTGF-treated MSCs were alpha-SMA-, differentiating into alpha-SMA+ myofibroblasts only when stimulated subsequently with TGF-beta1, suggestive of stepwise processes of fibroblast commitment, fibrogenesis, and pathological fibrosis. In rats, in vivo microencapsulated delivery of CTGF prompted postnatal connective tissue to undergo fibrogenesis rather than ectopic mineralization. The knowledge that fibroblasts have a mesenchymal origin may enrich our understanding of organ fibrosis, cancer stroma, ectopic mineralization, scarring, and regeneration.

Hybrid adipogenic implants from adipose stem cells for soft tissue reconstruction in vivo.

A critical barrier in tissue regeneration is scale-up. Bioengineered adipose tissue implants have been limited to ∼10 mm in diameter. Here, we devised a 40-mm hybrid implant with a cellular layer encapsulating an acellular core. Human adipose-derived stem cells (ASCs) were seeded in alginate. Poly(ethylene)glycol-diacrylate (PEGDA) was photopolymerized into 40-mm-diameter dome-shaped gel. Alginate-ASC suspension was painted onto PEGDA surface. Cultivation of hybrid constructs ex vivo in adipogenic medium for 28 days showed no delamination. Upon 4-week in vivo implantation in athymic rats, hybrid implants well integrated with host subcutaneous tissue and could only be surgically separated. Vascularized adipose tissue regenerated in the thin, painted alginate layer only if ASC-derived adipogenic cells were delivered. Contrastingly, abundant fibrous tissue filled ASC-free alginate layer encapsulating the acellular PEGDA core in control implants. Human-specific peroxisome proliferator-activated receptor-γ (PPAR-γ) was detected in human ASC-seeded implants. Interestingly, rat-specific PPAR-γ was absent in either human ASC-seeded or ASC-free implants. Glycerol content in ASC-delivered implants was significantly greater than that in ASC-free implants. Remarkably, rat-specific platelet/endothelial cell adhesion molecule (PECAM) was detected in both ASC-seeded and ASC-free implants, suggesting anastomosis of vasculature in bioengineered tissue with host blood vessels. Human nuclear staining revealed that a substantial number of adipocytes were of human origin, whereas endothelial cells of vascular wall were of chemaric human and nonhuman (rat host) origins. Together, hybrid implant appears to be a viable scale-up approach with volumetric retention attributable primarily to the acellular biomaterial core, and yet has a biologically viable cellular interface with the host. The present 40-mm soft tissue implant may serve as a biomaterial tissue expander for reconstruction of lumpectomy defects.

Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing.

Tooth infections or injuries involving dental pulp are treated routinely by root canal therapy. Endodontically treated teeth are devitalized, susceptible to re-infections, fractures, and subsequent tooth loss. Here, we report regeneration of dental-pulp-like tissue by cell homing and without cell transplantation. Upon in vivo implantation of endodontically treated real-size, native human teeth in mouse dorsum for the tested 3 weeks, delivery of basic fibroblast growth factor and/or vascular endothelial growth factor (bFGF and/or VEGF) yielded re-cellularized and revascularized connective tissue that integrated to native dentinal wall in root canals. Further, combined delivery of bFGF, VEGF, or platelet-derived growth factor (PDGF) with a basal set of nerve growth factor (NGF) and bone morphogenetic protein-7 (BMP7) generated cellularized and vascularized tissues positive of VEGF antibody staining and apparent neo-dentin formation over the surface of native dentinal wall in some, but not all, endodontically treated teeth. Newly formed dental pulp tissue appeared dense with disconnected cells surrounded by extracellular matrix. Erythrocyte-filled blood vessels were present with endothelial-like cell lining. Reconstructed, multiple microscopic images showed complete fill of dental-pulp-like tissue in the entire root canal from root apex to pulp chamber with tissue integration to dentinal wall upon delivery of bFGF, VEGF, or PDGF with a basal set of NGF and BMP7. Quantitative ELISA showed that combinatory delivery of bFGF, VEGF, or PDGF with basal NGF and BMP7 elaborated von Willerbrand factor, dentin sialoprotein, and NGF. These findings represent the first demonstration of regenerated dental-pulp-like tissue in endodontically treated root canals of real-size, native human teeth. The present chemotaxis-based approach has potent cell homing effects for re-cellularization and revascularization in endodontically treated root canals in vivo, although in an ectopic model. Regeneration of dental pulp by cell homing, rather than cell delivery, may accelerate clinical translation.

Facial reconstruction by biosurgery: cell transplantation versus cell homing.

The face distinguishes one human being from another. When the face is disfigured because of trauma, tumor removal, congenital anomalies, or chronic diseases, the patient has a strong desire for functional and esthetic restoration. Current practice of facial reconstruction using autologous grafts, synthetic fillers, and prostheses is frequently below the surgeon's and patient's expectations. Facial reconstruction is yet to take advantage of recent advances in seemingly unrelated fields of stem cell biology, chemical engineering, biomaterials, and tissue engineering. "Biosurgery," a new concept that we propose, will incorporate novel principles and strategies of bioactive cues, biopolymers, and/or cells to restore facial defects. Small facial defects can likely be reconstructed by cell homing and without cell transplantation. A critical advantage of cell homing is that agilely recruited endogenous cells have the potential to harness the host's innate capacity for regeneration, thus accelerating the rate of regulatory and commercialization processes for product development. Large facial defects, however, may not be restorable without cell delivery per our understanding at this time. New breakthrough in biosurgery will likely originate from integrated strategies of cell biology, cytokine biology, chemical engineering, biomaterials, and tissue engineering. Regardless of cell homing or cell delivery approaches, biosurgery not only will minimize surgical trauma and repetitive procedures, but also produce long-lasting results. At the same time, caution must be exercised against the development of products that lack scientific basis or dogmatic combination of cells, biomaterials, and biomolecules. Together, scientifically derived biosurgery will undoubtedly develop into new technologies that offer increasingly natural reconstruction and/or augmentation of the face.

Synergistic actions of hematopoietic and mesenchymal stem/progenitor cells in vascularizing bioengineered tissues.

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34(+) hematopoietic cells. Co-transplantation of CD34(+) HSCs and CD34(-) MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromised mice yielded vascularized tissue. The average vascular number of co-transplanted CD34(+) and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34(+) cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34(+) cells. Based on additional in vitro results of endothelial differentiation of CD34(+) cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34(+) and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34(+) cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.

Inhibition of osteogenic differentiation of human mesenchymal stem cells.

Mesenchymal stem cells (hMSCs) have been shown to differentiate into osteoblasts that, in turn, are capable of forming tissues analogous to bone. The present study was designed to investigate the inhibition of osteogenesis by hMSCs. Bone marrow-derived hMSCs were treated with transforming growth factor beta-3 (TGFbeta3) at various doses during or after their differentiation into osteogenic cells. TGFbeta3 was encapsulated in poly(DL-lactic-co-glycolic acid) (PLGA) microspheres and released via controlled delivery in the osteogenic culture of hMSCs and hMSC-derived osteoblasts for up to 28 days. Controlled release of TGFbeta3 inhibited the osteogenic differentiation of hMSCs, as evidenced by significantly reduced alkaline phosphatase activity and staining, as well as decreased mineral deposition. After hMSCs had been differentiated into osteoblasts, controlled release of TGFbeta3 further inhibited not only alkaline phosphatase and mineral deposition but also osteocalcin expression. These findings demonstrate the potential for sustained modulation of the behavior of stem cells and/or stem cell-derived lineage-specific cells via controlled release of growth factor(s). The attenuation of osteogenic differentiation of MSCs may facilitate understanding not only the regulation and patterning of osteogenesis in development but also several pathological models such as osteopetrosis, craniosynostosis, and heart valve calcification.

Labeling of mesenchymal stem cells by bioconjugated quantum dots.

Long-term labeling of stem cells during self-replication and differentiation benefits investigations of development and tissue regeneration. We report the labeling of human mesenchymal stem cells (hMSCs) with RGD-conjugated quantum dots (QDs) during self-replication, and multilineage differentiations into osteogenic, chondrogenic, and adipogenic cells. QD-labeled hMSCs remained viable as unlabeled hMSCs from the same subpopulation. These findings suggest the use of bioconjugated QDs as an effective probe for long-term labeling of stem cells.

Chondrogenesis of mesenchymal stem cells by controlled delivery of transforming growth factor-beta3.

Human mesenchymal stem cells (hMSC) have been shown to differentiate into chondrocytes and form cartilage-like tissues when cultured with TGFbeta3 at 10 ng/ml. Previous attempts to engineer cartilage using hMSC have depended on in vitro pre-differentiation in order to form chondrogenic engineered constructs. Such techniques greatly increase the time of implant fabrication and suffer from loss of phenotype upon withdrawal from chondrogenic medium. The present study investigates a tissue engineered construct that includes sustained delivery of TGFbeta3 and induces differentiation of hMSC into chondrocytes in situ using injectable thermosensitive gels.