Lessons from developmental biology for regenerative medicine.

The ultimate goal of regenerative medicine is the functional restoration of lost or damaged tissues and organs. Since most tissues in man lack true regenerative properties and instead respond to injury with an inflammatory response and scar tissue formation, regenerative medicine strategies that include combinations of cells, scaffolds, and bioactive molecules to replace injured or missing tissues have been developed. The physical, chemical, and electrical cues that define the microenvironmental niche and the effect of these influences upon cell behavior during development are of interest to developmental biologists, with obvious overlap to the interest of the regenerative medicine field. This manuscript provides an overview of current approaches for tissue restoration being investigated in the field of regenerative medicine and attempts to identify areas of mutual beneficial interest with the field of developmental biology.

Biologic scaffolds for musculotendinous tissue repair.

Traumatic injuries to the musculoskeletal system are common events and volumetric muscle loss (VML) is no longer a rare occurrence. Surgical intervention is typically the only option for restoration of partial function. Surgical intervention for VML however does not regenerate the lost tissue and typically results in alterations of both the anatomy and biomechanics at the site of injury. Non-traditional approaches to the restoration of functional musculoskeletal tissue, including those provided by tissue engineering and regenerative medicine strategies, become viable alternative therapies when the expected outcome is bleak. One such strategy involves the delivery of constructive cues and modulation of the micro-environmental niche via biologic scaffold materials. These materials ideally retain the native structure and composition of the extracellular matrix of the tissue from which they are derived. Some of the recent advances in the use of biologic scaffolds to target key stages of the musculotendinous repair process and promote the restoration of functional tissue are described herein.

Human NELL1 protein augments constructive tissue remodeling with biologic scaffolds.

Biologic scaffolds composed of extracellular matrix (ECM) derived from decellularized tissues effectively reprogram key stages of the mammalian response to injury, altering the wound microenvironment from one that promotes scar tissue formation to one that stimulates constructive and functional tissue remodeling. In contrast, engineered scaffolds, composed of purified ECM components such as collagen, lack the complex ultrastructure and composition of intact ECM and may promote wound healing but lack factors that facilitate constructive and functional tissue remodeling. The objective of the present study was to test the hypothesis that addition of NELL1, a signaling protein that controls cell growth and differentiation, enhances the constructive tissue remodeling of a purified collagen scaffold. An abdominal wall defect model in the rat of 1.5-cm(2) partial thickness was used to compare the constructive remodeling of a bovine type I collagen scaffold to a biologic scaffold derived from small intestinal submucosa (SIS)-ECM with and without augmentation with 17 µg NELL1 protein. Samples were evaluated histologically at 14 days and 4 months. The contractile response of the defect site was also evaluated at 4 months. Addition of NELL1 protein improved the constructive remodeling of collagen scaffolds but not SIS-ECM scaffolds. Results showed an increase in the contractile force of the remodeled skeletal muscle and a fast:slow muscle composition similar to native tissue in the collagen-treated group. The already robust remodeling response to SIS-ECM was not enhanced by NELL1 at the dose tested. These findings suggest that NELL1 protein does contribute to the enhanced constructive remodeling of skeletal muscle.

Bone marrow-derived cells participate in the long-term remodeling in a mouse model of esophageal reconstruction.

BACKGROUND: The default response of the esophagus to injury includes inflammation and scar tissue formation often leading to stricture. Biologic scaffolds composed of extracellular matrix (ECM) have been associated with the reconstitution of functional esophageal tissue in preclinical studies and clinical case reports of esophageal mucosal resection, anastomotic reinforcement, and full circumferential replacement. However, the mechanisms responsible for this change in the default response to esophageal injury are not fully understood. METHODS: The objective of the present study was to determine whether bone marrow-derived cells (BMCs) participate in the long-term remodeling of ECM scaffolds in the esophageal location in a mouse model. RESULTS: BMCs were present in low numbers in remodeling ECM scaffolds. Compared with the untreated control mice, the ECM-implanted animals showed better remodeling of the epithelial layer. CONCLUSIONS: BMCs are involved in ECM remodeling process during tissue repair after esophageal injury, but the low numbers argue against any significant involvement in the constructive remodeling process.

Regeneration of skeletal muscle.

Skeletal muscle has a robust capacity for regeneration following injury. However, few if any effective therapeutic options for volumetric muscle loss are available. Autologous muscle grafts or muscle transposition represent possible salvage procedures for the restoration of mass and function but these approaches have limited success and are plagued by associated donor site morbidity. Cell-based therapies are in their infancy and, to date, have largely focused on hereditary disorders such as Duchenne muscular dystrophy. An unequivocal need exists for regenerative medicine strategies that can enhance or induce de novo formation of functional skeletal muscle as a treatment for congenital absence or traumatic loss of tissue. In this review, the three stages of skeletal muscle regeneration and the potential pitfalls in the development of regenerative medicine strategies for the restoration of functional skeletal muscle in situ are discussed.

Biologic scaffold remodeling in a dog model of complex musculoskeletal injury.

BACKGROUND: Current treatment principles for muscle injuries with volumetric loss have been largely derived from empirical observations. Differences in severity or anatomic location have determinant effects on the tissue remodeling outcome. Biologic scaffolds composed of extracellular matrix (ECM) have been successfully used to restore vascularized, innervated, and contractile skeletal muscle in animal models but limited anatomic locations have been evaluated. The aim of this study was to determine the ability of a xenogeneic ECM scaffold to restore functional skeletal muscle in a canine model of a complex quadriceps injury involving bone, tendon, and muscle. MATERIALS AND METHODS: Sixteen dogs were subjected to unilateral resection of the distal third of the vastus lateralis and medial half of the distal third of the vastus medialis muscles including the proximal half of their associated quadriceps tendon. This defect was replaced with a biologic scaffold composed of small intestinal submucosa extracellular matrix (SIS-ECM) and the remodeling response was evaluated at 1, 2, 3, and 6 mo (N = 4 per group). RESULTS: The initial remodeling process followed a similar pattern to other studies of ECM-mediated muscle repair with rapid vascularization and migration of myoblasts into the defect site. However, over time the remodeling response resulted in the formation of dense collagenous tissue with islands of muscle in the segments of the scaffold not in contact with bone, and foci of bone and cartilage in the segments that were adjacent to the underlying bone. CONCLUSIONS: SIS-ECM was not successful at restoring functional muscle tissue in this model. However, the results also suggest that SIS-ECM may have potential to promote integration of soft and boney tissues when implanted in close apposition to bone.

Matrix Bound Nanovesicles Have Tissue-Specific Characteristics That Suggest a Regulatory Role.

Recent studies have identified an extracellular vesicle population that is tightly anchored within the extracellular matrix (ECM) of tissues and organs until released by matrix turnover events. Evidence suggests that these matrix-bound nanovesicles (MBVs) are a ubiquitous component of the ECM, raising questions regarding their tissue-specific identity and their biologic function(s). The primary objective of this study was to examine MBVs isolated from six different tissues and compare their physical and compositional characteristics to determine the common and differentially expressed features. Accordingly, the results of this characterization show that while MBVs are a ubiquitous component of the ECM, they contain a protein and microRNA cargo that is tissue specific. The results furthermore suggest that MBVs have an important role in regulating tissue homeostasis.

A histomorphologic study of the normal healing response following digit amputation in C57bl/6 and MRL/MpJ mice.

Mice are common models for the study of mammalian wound healing. However, the array of available phenotypes suggests that significant differences likely exist in the normal wound healing response between different mouse strains. It is therefore essential to understand the normal healing response for each mouse strain, anatomic site, and mechanism of injury when investigating the potential effects of therapeutic interventions upon the healing response. The objective of the present study was to characterize and compare the morphologic changes that occur in both the MRL/MpJ and C57bl/6 mice strains during the first 14 days following amputation at the midpoint of the second phalanx. Our results identify noticeable temporal and spatial differences between the two strains, particularly in the expression of CD34+ and CD133+ progenitor cells, the re-epithelialization of the wound and deposition of type I and type III collagen. Unlike other selected tissues in which MRL/MpJ mice demonstrate a capacity to completely regenerate lost tissue, the responses observed in this model of digit healing did not translate into a greater capacity to regenerate lost structures. Both mouse strains show a similar healing response by day 14.

Matrix-Bound Nanovesicles: The Effects of Isolation Method upon Yield, Purity, and Function.

Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) raises questions regarding their biologic functions and their potential theranostic application. Unlike liquid-phase extracellular vesicles (e.g., exosomes), MBV are tightly bound to the ECM, which makes their isolation and harvesting more challenging. The indiscriminate use of different methods to harvest MBV can alter or disrupt their structural and/or functional integrity. The objective of the present study was to compare the effect of various MBV harvesting methods upon yield, purity, and biologic activity. Combinations of four methods to solubilize the ECM (collagenase [COL], liberase [LIB], or proteinase K [PK] and nonenzymatic elution with potassium chloride) and four isolation methods (ultracentrifugation, ultrafiltration [UF], density barrier, and size exclusion chromatography [SEC]) were used to isolate MBV from urinary bladder-derived ECM. All combinations of solubilization and isolation methods allowed for the harvesting of MBV, however, distinct differences were noted. The highest yield, purity, cellular uptake, and biologic activity were seen with MBV isolated by a combination of liberase or collagenase followed by SEC. The combination of proteinase K and UF was shown to have detrimental effects on bioactivity. The results show the importance of selecting appropriate MBV harvesting methods for the characterization and evaluation of MBV and for analysis of their potential theranostic application. Impact statement Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) has raised questions regarding their biologic functions and their potential theranostic application. This study demonstrates that the harvesting methods used can result in samples with physical and biochemical properties that are unique to the isolation and solubilization methods used. Consequently, developing harvesting methods that minimize sample contamination with ECM remnants and/or solubilization agents will be essential in determining the theranostic potential of MBV in future studies.

4-Hydroxybutyrate Promotes Endogenous Antimicrobial Peptide Expression in Macrophages.

IMPACT STATEMENT: This study evaluated the biological activity of hydroxylated derivatives of butyrate as inductors of antimicrobial peptides (AMPs) in murine bone marrow-derived macrophages in vitro. A differential modulation of AMP expression by the hydroxylated derivatives of butyrate is shown. The ability of sodium 4-hydroxybutyrate to upregulate AMP expression through a histone deacetylase inhibitory-independent mechanism, and to promote increased resistance to bacterial contamination in vivo are also shown. The findings provide an alternative for prevention of bacterial contamination of implanted biomaterials. Functionalization of biomaterials with hydroxylated derivatives of butyrate can enhance the endogenous antimicrobial activity of the immune system through increased production of AMPs by host cells, thus providing protection against bacterial contamination.

Extracellular Matrix Degradation Products Downregulate Neoplastic Esophageal Cell Phenotype.

IMPACT STATEMENT: Extracellular matrix (ECM) biomaterials were used to treat esophageal cancer patients after cancer resection and promoted regrowth of normal mucosa without recurrence of cancer. The present study investigates the mechanisms by which these materials were successful to prevent the cancerous phenotype. ECM downregulated neoplastic esophageal cell function (proliferation, metabolism), but normal esophageal epithelial cells were unaffected in vitro, and suggests a molecular basis (downregulation of PI3K-Akt, cell cycle) for the promising clinical results. The therapeutic effect appeared to be enhanced using homologous esophageal ECM. This study suggests that ECM can be further investigated to treat cancer patients after resection or in combination with targeted therapy.

Cyclic stretch-induced TGFbeta1/Smad signaling inhibits adipogenesis in umbilical cord progenitor cells.

Human umbilical cord perivascular cells (HUCPVCs) can differentiate along numerous lineages making them a favourable cell source for tissue regeneration. However, how these cells respond to biomechanical forces is unclear. This study aimed to determine whether cyclic stretch could regulate adipogenic differentiation of HUCPVCs, and to elucidate the mechanism of this regulation. In adipogenic culture, HUCPVCs expressed the adipocyte-specific transcription factors PPARgamma and C/EBPalpha and accumulated cytoplasmic lipid droplets. Exposure of these cells to equibiaxial cyclic stretch (10%, 0.5 Hz) in the presence of adipogenic medium, increased Smad2 phosphorylation compared to static samples and inhibited the expression of adipocyte markers; ERK1/2 phosphorylation was not changed. Inhibiting TGFbeta1 signaling decreased Smad2 phosphorylation and prevented the inhibition of adipogenic differentiation by cyclic stretch. These results demonstrate that cyclic equibiaxial stretch regulates HUCPVC differentiation even in the presence of an adipogenic milieu and should be an important consideration in developing future progenitor cell therapies.

The effects of stretch on vascular smooth muscle cell phenotype in vitro.

Vascular smooth muscle cells (VSMC) situated in the tunica media of veins and arteries are central to maintaining conduit integrity in the face of mechanical forces inherent within the cardiovascular system. The predominant mechanical force influencing VSMC structural organisation and signalling is cyclic stretch. VSMC phenotype is manipulated by externally applied stretch which regulates the activity of their contractile apparatus. Stretch modulates cell shape, cytoplasmic organisation, and intracellular processes leading to migration, proliferation, or contraction. Drug therapy directed at the components of the signalling pathways influenced by stretch may ultimately prevent cardiovascular pathology such as myointimal hyperplasia.

Sutureless nerve repair with ECM bioscaffolds and laser-activated chitosan adhesive.

The outcome of peripheral nerve repair following transection is influenced by a number of factors but almost all approaches require anastomosis of the nerve using technically demanding microsurgical procedures. However, the use of sutures presents a number of unavoidable challenges including additional nerve trauma, stimulation of an inflammatory response, and endoneural fibrosis. The objective of the present study was to determine the efficacy of a sutureless approach to nerve repair. A rat sciatic nerve transection model was used with a laser-activated, chitosan-based adhesive (SurgiLux), combined with different forms of extracellular matrix (ECM), known to promote Schwann cell proliferation and nerve growth both in peripheral nerve applications. Following a 5 mm transection of the sciatic nerve, nerve guide wraps were prepared using: (1) laser-activated adhesive (SurgiLux) alone, (2) SurgiLux incorporating ECM (SurgiLux ECM), (3) ECM secured using SurgiLux, and (4) ECM secured using 8-0 Prolene sutures. A no treatment groups was used as a negative control. Evaluation of tissue remodeling was conducted with histolomorphometric assessment of neuroma, integrity of repair, nerve immunolabeling, ratio of myelinated to non-myelinated fibers, and amount of connective tissue. Quantitative and semi-quantitative analysis of the repaired nerve transections at 6 and 12 weeks showed that that SurgiLux incorporating powdered ECM (SurgiLux ECM), SurgiLux alone and ECM alone all improved the healing response compared to no-treatment controls, with less fibrotic tissue and more nerve staining. Histologic scoring showed that the SurgiLux ECM group showed the greatest increase in histologic score between the two time points tested. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1698-1711, 2018.

Alpha2(VIII) collagen substrata enhance endothelial cell retention under acute shear stress flow via an alpha2beta1 integrin-dependent mechanism: an in vitro and in vivo study.

BACKGROUND: Essential to tissue-engineered vascular grafts is the formation of a functional endothelial monolayer capable of resisting the forces of blood flow. This study targeted alpha2(VIII) collagen, a major component of the subendothelial matrix, and examined the ability of and mechanisms by which endothelial cells attach to this collagen under static and dynamic conditions both in vitro and in vivo. METHODS AND RESULTS: Attachment of human endothelial cells to recombinant alpha2(VIII) collagen was assessed in vitro under static and shear conditions of up to 100 dyne/cm2. Alpha2(VIII) collagen supported endothelial cell attachment in a dose-dependent manner, with an 18-fold higher affinity for endothelial cells compared with fibronectin. Cell attachment was significantly inhibited by function-blocking anti-alpha2 (56%) and -beta1 (98%) integrin antibodies but was not RGD dependent. Under flow, endothelial cells were retained at significantly higher levels on alpha2(VIII) collagen (53% and 51%) than either fibronectin (23% and 16%) or glass substrata (7% and 1%) at shear rates of 30 and 60 dyne/cm2, respectively. In vivo studies, using endothelialized polyurethane grafts, demonstrated significantly higher cell retention rates to alpha2(VIII) collagen-coated than to fibronectin-coated prostheses in the midgraft area (P < 0.05) after 24 hours' implantation in the caprine carotid artery. CONCLUSIONS: These studies demonstrate that alpha2(VIII) collagen has the potential to improve both initial cell attachment and retention of endothelial cells on vascular grafts in vivo, which opens new avenues of research into the development of single-stage endothelialized prostheses and the next generation of tissue-engineered vascular grafts.

The effect of cell debris within biologic scaffolds upon the macrophage response.

All biomaterials, including biologic scaffolds composed of extracellular matrix (ECM), elicit a host immune response when implanted. The type and intensity of this response depends in part upon the thoroughness of decellularization and removal of cell debris from the source tissue. Proinflammatory responses have been associated with negative downstream remodeling events including scar tissue formation, encapsulation, and seroma formation. The relative effects of specific cellular components upon the inflammatory response are not known. The objective of the present study was to determine the effect of different cell remnants that may be present in ECM scaffold materials upon the host innate immune response, both in vitro and in vivo. Collagen scaffolds were supplemented with one of three different concentrations of DNA, mitochondria, or cell membranes. Murine macrophages were exposed to the various supplemented scaffolds and the effect upon macrophage phenotype was evaluated. In vivo studies were performed using an abdominal wall defect model in the rat to evaluate the effect of the scaffolds upon the macrophage response. Murine macrophages exposed in vitro to scaffolds supplemented with DNA, mitochondria, and cell membranes showed increased expression of proinflammatory M1 marker iNOS and no expression of the proremodeling M2 marker Fizz1 regardless of supplementation concentration. A dose-dependent response was observed in the rat model for collagen scaffolds supplemented with cell remnants. DNA, mitochondria, and cell membrane remnants in collagen scaffolds promote a proinflammatory M1 macrophage phenotype in vivo and in vitro. These results reinforce the importance of a thorough decellularization process for ECM biologic scaffold materials. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2109-2118, 2017.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. STATEMENT OF SIGNIFICANCE: Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.

The impact of detergents on the tissue decellularization process: A ToF-SIMS study.

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy. STATEMENT OF SIGNIFICANCE: We report here on the use of a highly sensitive analytical technique, time of flight secondary ion mass spectroscopy (ToF-SIMS) to characterize detergent decellularized scaffolds. ToF-SIMS detected cellular remnants and residual detergent fragments; increased intensity of the detergent fragments correlated with adverse cell matrix interactions. This study demonstrates the importance of maintaining a balance between cell removal and detergent disruption of matrix architecture and matrix surface ligand landscape. This study also demonstrates the power of ToF-SIMS for the characterization of decellularized scaffolds and capability for assessment of decellularization efficacy. Future use of biologic scaffolds in clinical tissue reconstruction will benefit from the fundamental results described in this work.

Upper dorsal endoscopic thoracic sympathectomy: a comparison of one- and two-port ablation techniques.

OBJECTIVE: Facial blushing and hyperhidrosis, particularly in the facial, axillary or palmar distribution, are socially, professionally, and psychologically debilitating conditions. Endoscopic thoracic sympathectomy can be carried out through multiple ports or by using a single port and a modified thoracoscope with integrated electrocautery. We reviewed our own experience to compare outcomes between these methods. METHODS: One hundred and nine consecutive endoscopic thoracic sympathectomies performed on 96 patients (M:F, 30:66) were examined with respect to operative method, symptom control, and patient satisfaction. Complete follow-up was available on 144 treated sides in 77 patients (80.2%), 38 treated with two ports, 39 performed by a one-port procedure. Mean age was 32.6 years (range 18-63) with a median follow-up of 25 months (range 5-85). Pooled data showed that the mean duration hospital stay was 1.6 nights with no deaths, conversions, or neurological injuries. RESULTS: The one-port group showed superior outcomes in terms of hospital stay, rate of postoperative pneumothorax, and the need for chest drain insertion; however, there was no correlation between number of ports and patient satisfaction. The mean overall satisfaction rating out of 5 was 3.3 with 76.6% of patients rating the outcome as 3 or more. 90.9% had an initial improvement in symptoms, although 21 patients (27.3%) described a late return of symptoms. CONCLUSION: Endoscopic thoracic sympathectomy can be safely and effectively carried out using a single port with similar results to the traditional two-port procedure. The one-port procedure may allow for a shorter duration of stay and lower complication rate.

A panel data set on harvest and perfusion decellularization of porcine rectus abdominis.

In this dataset, we particularly depicted the harvest and perfusion decellularization of porcine rectus abdominis (RA), accompanied with displaying of the retained vascular trees within the perfusion-decellularized skeletal muscle matrix (pM-ECM) using vascular corrosion casting. In addition, several important tips for successful pM-ECM preparation were emphasized, which including using anatomically isolated skeletal muscle as tissue source with all main feeding and draining vessels perfused, preserving the internal microcirculation availability, aseptic technique and pyrogen free in all steps, sequential perfusion via artery or vein, and longtime washing after decellularization. The data are supplemental to our original research article describing detailed associations of pM-ECM as a clinically relevant scale, three-dimensional scaffold with a vascular network template for tissue-specific regeneration, "Perfusion-decellularized skeletal muscle as a three-dimensional scaffold with a vascular network template" Zhang et al. (2016) [1].