Porcine Ischemic Wound-Healing Model for Preclinical Testing of Degradable Biomaterials.

Impaired wound healing that mimics chronic human skin pathologies is difficult to achieve in current animal models, hindering testing and development of new therapeutic biomaterials that promote wound healing. In this article, we describe a refinement and simplification of the porcine ischemic wound model that increases the size and number of experimental sites per animal. By comparing three flap geometries, we adopted a superior configuration (15 × 10 cm) that enabled testing of twenty 1 cm2 wounds in each animal: 8 total ischemic wounds within 4 bipedicle flaps and 12 nonischemic wounds. The ischemic wounds exhibited impaired skin perfusion for ∼1 week. To demonstrate the utility of the model for comparative testing of tissue regenerative biomaterials, we evaluated the healing process in wounds implanted with highly porous poly (thioketal) urethane (PTK-UR) scaffolds that were fabricated through reaction of reactive oxygen species (ROS)-cleavable PTK macrodiols with isocyanates. PTK-lysine triisocyanate (LTI) scaffolds degraded significantly in vitro under both oxidative and hydrolytic conditions whereas PTK-hexamethylene diisocyanate trimer (HDIt) scaffolds were resistant to hydrolytic breakdown and degraded exclusively through an ROS-dependent mechanism. Upon placement into porcine wounds, both types of PTK-UR materials fostered new tissue ingrowth over 10 days in both ischemic and nonischemic tissue. However, wound perfusion, tissue infiltration and the abundance of pro-regenerative, M2-polarized macrophages were markedly lower in ischemic wounds independent of scaffold type. The PTK-LTI implants significantly improved tissue infiltration and perfusion compared with analogous PTK-HDIt scaffolds in ischemic wounds. Both LTI and HDIt-based PTK-UR implants enhanced M2 macrophage activity, and these cells were selectively localized at the scaffold/tissue interface. In sum, this modified porcine wound-healing model decreased animal usage, simplified procedures, and permitted a more robust evaluation of tissue engineering materials in preclinical wound healing research. Deployment of the model for a relevant biomaterial comparison yielded results that support the use of the PTK-LTI over the PTK-HDIt scaffold formulation for future advanced therapeutic studies.

Local Delivery of PHD2 siRNA from ROS-Degradable Scaffolds to Promote Diabetic Wound Healing.

Small interfering RNA (siRNA) delivered from reactive oxygen species-degradable tissue engineering scaffolds promotes diabetic wound healing in rats. Porous poly(thioketal-urethane) scaffolds implanted in diabetic wounds locally deliver siRNA that inhibits the expression of prolyl hydroxylase domain protein 2, thereby increasing the expression of progrowth genes and increasing vasculature, proliferating cells, and tissue development in diabetic wounds.

BMP1-like proteinases are essential to the structure and wound healing of skin.

Closely related extracellular metalloproteinases bone morphogenetic protein 1 (BMP1) and mammalian Tolloid-like 1 (mTLL1) are co-expressed in various tissues and have been suggested to have overlapping roles in the biosynthetic processing of extracellular matrix components. Early lethality of mice null for the BMP1 gene Bmp1 or the mTLL1 gene Tll1 has impaired in vivo studies of these proteinases. To overcome issues of early lethality and functional redundancy we developed the novel BTKO mouse strain, with floxed Bmp1 and Tll1 alleles, for induction of postnatal, simultaneous ablation of the two genes. We previously showed these mice to have a skeletal phenotype that includes elements of osteogenesis imperfecta (OI), osteomalacia, and deficient osteocyte maturation, observations validated by the finding of BMP1 mutations in a subset of human patients with OI-like phenotypes. However, the roles of BMP1-like proteinase in non-skeletal tissues have yet to be explored, despite the supposed importance of putative substrates of these proteinases in such tissues. Here, we employ BTKO mice to investigate potential roles for these proteinases in skin. Loss of BMP1-like proteinase activity is shown to result in markedly thinned and fragile skin with unusually densely packed collagen fibrils and delayed wound healing. We demonstrate deficits in the processing of collagens I and III, decorin, biglycan, and laminin 332 in skin, which indicate mechanisms whereby BMP1-like proteinases affect the biology of this tissue. In contrast, lack of effects on collagen VII processing or deposition indicates this putative substrate to be biosynthetically processed by non-BMP1-like proteinases.

Wound samples: moving towards a standardised method of collection and analysis.

Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, impact the lives of millions of people worldwide. These types of wounds represent a significant physical, social and financial burden to both patients and health care systems. Wound care has made great progress in recent years as a result of the critical research performed in academic, clinical and industrial settings. However, there has been relatively little translation of basic research discoveries into novel and effective treatments. One underlying reason for this paucity may be inconsistency in the methods of wound analysis and sample collection, resulting in the inability of researchers to accurately characterise the healing process and compare results from different studies. This review examines the various types of analytical methods being used in wound research today with emphasis on sampling techniques, processing and storage, and the findings call forth the wound care research community to standardise its approach to wound analysis in order to yield more robust and comparable data sets.

A transient cell-shielding method for viable MSC delivery within hydrophobic scaffolds polymerized in situ.

Cell-based therapies have emerged as promising approaches for regenerative medicine. Hydrophobic poly(ester urethane)s offer the advantages of robust mechanical properties, cell attachment without the use of peptides, and controlled degradation by oxidative and hydrolytic mechanisms. However, the application of injectable hydrophobic polymers to cell delivery is limited by the challenges of protecting cells from reaction products and creating a macroporous architecture post-cure. We designed injectable carriers for cell delivery derived from reactive, hydrophobic polyisocyanate and polyester triol precursors. To overcome cell death caused by reaction products from in situ polymerization, we encapsulated bone marrow-derived stem cells (BMSCs) in fastdegrading, oxidized alginate beads prior to mixing with the hydrophobic precursors. Cells survived the polymerization at >70% viability, and rapid dissolution of oxidized alginate beads after the scaffold cured created interconnected macropores that facilitated cellular adhesion to the scaffold in vitro. Applying this injectable system to deliver BMSCs to rat excisional skin wounds showed that the scaffolds supported survival of transplanted cells and infiltration of host cells, which improved new tissue formation compared to both implanted, pre-formed scaffolds seeded with cells and acellular controls. Our design is the first to enable injectable delivery of settable, hydrophobic scaffolds where cell encapsulation provides a mechanism for both temporary cytoprotection during polymerization and rapid formation of macropores post-polymerization. This simple approach provides potential advantages for cell delivery relative to hydrogel technologies, which have weaker mechanical properties and require incorporation of peptides to achieve cell adhesion and degradability.

Global deletion of Ankrd1 results in a wound-healing phenotype associated with dermal fibroblast dysfunction.

The expression of ankyrin repeat domain protein 1 (Ankrd1), a transcriptional cofactor and sarcomeric component, is strongly elevated by wounding and tissue injury. We developed a conditional Ankrd1(fl/fl) mouse, performed global deletion with Sox2-cre, and assessed the role of this protein in cutaneous wound healing. Although global deletion of Ankrd1 did not affect mouse viability or development, Ankrd1(-/-) mice had at least two significant wound-healing phenotypes: extensive necrosis of ischemic skin flaps, which was reversed by adenoviral expression of ANKRD1, and delayed excisional wound closure, which was characterized by decreased contraction and reduced granulation tissue thickness. Skin fibroblasts isolated from Ankrd1(-/-) mice did not spread or migrate on collagen- or fibronectin-coated surfaces as efficiently as fibroblasts isolated from Ankrd1(fl/fl) mice. More important, Ankrd1(-/-) fibroblasts failed to contract three-dimensional floating collagen gels. Reconstitution of ANKRD1 by adenoviral infection stimulated both collagen gel contraction and actin fiber organization. These in vitro data were consistent with in vivo wound closure studies, and suggest that ANKRD1 is important for the proper interaction of fibroblasts with a compliant collagenous matrix both in vitro and in vivo.

Enhanced Performance of Plasmid DNA Polyplexes Stabilized by a Combination of Core Hydrophobicity and Surface PEGylation.

Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability, endosomal escape, and resultant high transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))-pDNA polyplexes underscores their potential utility both for local delivery from scaffolds as well as systemic, intravenous delivery.

Biodegradable lysine-derived polyurethane scaffolds promote healing in a porcine full-thickness excisional wound model.

Lysine-derived polyurethane scaffolds (LTI-PUR) support cutaneous wound healing in loose-skinned small animal models. Due to the physiological and anatomical similarities of human and pig skin, we investigated the capacity of LTI-PUR scaffolds to support wound healing in a porcine excisional wound model. Modifications to scaffold design included the addition of carboxymethylcellulose (CMC) as a porogen to increase interconnectivity and an additional plasma treatment (Plasma) to decrease surface hydrophobicity. All LTI-PUR scaffold and formulations supported cellular infiltration and were biodegradable. At 15 days, CMC and plasma scaffolds simulated increased macrophages more so than LTI PUR or no treatment. This response was consistent with macrophage-mediated oxidative degradation of the lysine component of the scaffolds. Cell proliferation was similar in control and scaffold-treated wounds at 8 and 15 days. Neither apoptosis nor blood vessel area density showed significant differences in the presence of any of the scaffold variations compared with untreated wounds, providing further evidence that these synthetic biomaterials had no adverse effects on those pivotal wound healing processes. During the critical phase of granulation tissue formation in full thickness porcine excisional wounds, LTI-PUR scaffolds supported tissue infiltration, while undergoing biodegradation. Modifications to scaffold fabrication modify the reparative process. This study emphasizes the biocompatibility and favorable cellular responses of PUR scaffolding formulations in a clinically relevant animal model.

Biomolecular signatures of diabetic wound healing by structural mass spectrometry.

Wound fluid is a complex biological sample containing byproducts associated with the wound repair process. Contemporary techniques, such as immunoblotting and enzyme immunoassays, require extensive sample manipulation and do not permit the simultaneous analysis of multiple classes of biomolecular species. Structural mass spectrometry, implemented as ion mobility-mass spectrometry (IM-MS), comprises two sequential, gas-phase dispersion techniques well suited for the study of complex biological samples because of its ability to separate and simultaneously analyze multiple classes of biomolecules. As a model of diabetic wound healing, poly(vinyl alcohol) sponges were inserted subcutaneously into nondiabetic (control) and streptozotocin-induced diabetic rats to elicit a granulation tissue response and to collect acute wound fluid. Sponges were harvested at days 2 or 5 to capture different stages of the early wound-healing process. Utilizing IM-MS, statistical analysis, and targeted ultraperformance liquid chromatography analysis, biomolecular signatures of diabetic wound healing have been identified. The protein S100-A8 was highly enriched in the wound fluids collected from day 2 diabetic rats. Lysophosphatidylcholine (20:4) and cholic acid also contributed significantly to the differences between diabetic and control groups. This report provides a generalized workflow for wound fluid analysis demonstrated with a diabetic rat model.

A physiological role for connective tissue growth factor in early wound healing.

Mesenchymal stem cells (MSCs) that overexpress secreted frizzled-related protein 2 (sFRP2) exhibit an enhanced reparative phenotype. The secretomes of sFRP2-overexpressing MSCs and vector control-MSCs were compared through liquid chromatography tandem mass spectrometry. Proteomic profiling revealed that connective tissue growth factor (CTGF; CCN2) was overrepresented in the conditioned media of sFRP2-overexpressing MSCs and MSC-derived CTGF could thus be an important paracrine effector. Subcutaneously implanted, MSC-loaded polyvinyl alcohol (PVA) sponges and stented excisional wounds were used as wound models to study the dynamics of CTGF expression. Granulation tissue generated within the sponges and full-thickness skin wounds showed transient upregulation of CTGF expression by MSCs and fibroblasts, implying a role for this molecule in early tissue repair. Although collagen and COL1A2 mRNA were not increased when recombinant CTGF was administered to sponges during the early phase (day 1-6) of tissue repair, prolonged administration (>15 days) of exogenous CTGF into PVA sponges resulted in fibroblast proliferation and increased deposition of collagen within the experimental granulation tissue. In support of its physiological role, CTGF immunoinhibition during early repair (days 0-7) reduced the quantity, organizational quality and vascularity of experimental granulation tissue in the sponge model. However, CTGF haploinsufficiency was not enough to reduce collagen deposition in excisional wounds. Similar to acute murine wound models, CTGF was transiently present in the early phase of human acute burn wound healing. Together, these results further support a physiological role for CTGF in wound repair and demonstrate that when CTGF expression is confined to early tissue repair, it serves a pro-reparative role. These data also further illustrate the potential of MSC-derived paracrine modulators to enhance tissue repair.

Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.

In vivo analysis of laser preconditioning in incisional wound healing of wild-type and HSP70 knockout mice with Raman spectroscopy.

BACKGROUND AND OBJECTIVE: Laser preconditioning augments incisional wound healing by reducing scar tissue and increasing maximum tensile load of the healed wound [Wilmink et al. (2009) J Invest Dermatol 129(1): 205-216]. Recent studies have optimized treatments or confirmed results using HSP70 as a biomarker. Under the hypothesis that HSP70 plays a role in reported results and to better understand the downstream effects of laser preconditioning, this study utilized a probe-based Raman spectroscopy (RS) system to achieve an in vivo, spatio-temporal biochemical profile of murine skin incisional wounds as a function of laser preconditioning and the presence of HSP70. STUDY DESIGN/MATERIALS AND METHODS: A total of 19 wild-type (WT) and HSP70 knockout (HSP70-/-) C57BL/6 mice underwent normal and laser preconditioned incisional wounds. Laser thermal preconditioning was conducted via previously established protocol (λ = 1.85 µm, H(0 ) = 7.64 mJ/cm(2) per pulse, spot diameter = 5 mm, Rep. rate = 50 Hz, τ(p) = 2 milliseconds, exposure time = 10 minutes) with an Aculight Renoir diode laser, with tissue temperature confirmed by real-time infrared camera measurements. Wound-healing progression was quantified by daily collection of a spatial distribution of Raman spectra. The results of RS findings were then qualified using standard histology and polarization microscopy. RESULTS: Raman spectra yielded significant differences (t-test; α = 0.05) in several known biochemical peaks between WT and HSP70 (-/-) mice on wounds and in adjacent tissue early in the wound-healing process. Analysis of peak ratios implied (i) an increase in protein configuration in and surrounding the wound in WT mice, and (ii) an increased cellular trend in WT mice that was prolonged due to laser treatment. Polarization microscopy confirmed that laser treated WT mice showed increased heterogeneity in collagen orientation. CONCLUSIONS: The data herein supports the theory that HSP70 is involved in normal skin protein configuration and the cellularity of early wound healing. Laser preconditioning extends cellular trends in the presence of HSP70. Despite study limitations, RS provided a non-invasive method for quantifying temporal trends in altered wound healing, narrowing candidates and design for future studies with clinically applicable instrumentation.

Pivotal role for alpha1-antichymotrypsin in skin repair.

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.

Dynamic reciprocity in the wound microenvironment.

Here, we define dynamic reciprocity (DR) as an ongoing, bidirectional interaction among cells and their surrounding microenvironment. In this review, we posit that DR is especially meaningful during wound healing as the DR-driven biochemical, biophysical, and cellular responses to injury play pivotal roles in regulating tissue regenerative responses. Such cell-extracellular matrix interactions not only guide and regulate cellular morphology, but also cellular differentiation, migration, proliferation, and survival during tissue development, including, e.g., embryogenesis, angiogenesis, as well as during pathologic processes including cancer, diabetes, hypertension, and chronic wound healing. Herein, we examine DR within the wound microenvironment while considering specific examples across acute and chronic wound healing. This review also considers how a number of hypotheses that attempt to explain chronic wound pathophysiology may be understood within the DR framework. The implications of applying the principles of DR to optimize wound care practice and future development of innovative wound healing therapeutics are also briefly considered.

The effect of the local delivery of platelet-derived growth factor from reactive two-component polyurethane scaffolds on the healing in rat skin excisional wounds.

A key tenet of tissue engineering is the principle that the scaffold can perform the dual roles of biomechanical and biochemical support through presentation of the appropriate mediators to surrounding tissue. While growth factors have been incorporated into scaffolds to achieve sustained release, there are a limited number of studies investigating release of biologically active molecules from reactive two-component polymers, which have potential application as injectable delivery systems. In this study, we report the sustained release of platelet-derived growth factor (PDGF) from a reactive two-component polyurethane. The release of PDGF was bi-phasic, characterized by an initial burst followed by a period of sustained release for up to 21 days. Despite the potential for amine and hydroxyl groups in the protein to react with the isocyanate groups in the reactive polyurethane, the in vitro bioactivity of the released PDGF was largely preserved when added as a lyophilized powder. PUR/PDGF scaffolds implanted in rat skin excisional wounds accelerated wound healing relative to the blank PUR control, resulting in almost complete healing with reepithelization at day 14. The presence of PDGF attracted both fibroblasts and mononuclear cells, significantly accelerating degradation of the polymer and enhancing formation of new granulation tissue as early as day 3. The ability of reactive two-component PUR scaffolds to promote new tissue formation in vivo through local delivery of PDGF may present compelling opportunities for the development of novel injectable therapeutics.

Enhanced angiogenesis and reduced contraction in thrombospondin-2-null wounds is associated with increased levels of matrix metalloproteinases-2 and -9, and soluble VEGF.

Thrombospondin-2 (TSP2) is an inhibitor of angiogenesis with pro-apoptotic and anti-proliferative effects on endothelial cells. Mice deficient in this matricellular protein display improved recovery from ischemia and accelerated wound healing associated with alterations in angiogenesis and extracellular matrix remodeling. In this study, we probed the function of TSP2 by performing a detailed analysis of dermal wounds and wound-derived fibroblasts. Specifically, we analyzed incisional wounds by tensiometry and found no differences in strength recovery between wild-type and TSP2-null mice. In addition, analysis of full-thickness excisional wounds by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling stain and MIB-5 immunohistochemistry revealed similar numbers of apoptotic and proliferating cells, respectively. In contrast, the levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2, and soluble vascular endothelial growth factor were increased in wounds of TSP2-null mice. Evaluation of the ability of TSP2-null wound fibroblasts to contract collagen gels revealed that it was compromised, even though TSP2-null wounds displayed normal myofibroblast content. Therefore, we conclude that the lack of TSP2 leads to aberrant extracellular matrix remodeling, increased neovascularization, and reduced contraction due in part to elevated levels of MMP-2 and MMP-9. These observations provide in vivo supporting evidence for a newly proposed function of TSP2 as a modulator of extracellular matrix remodeling.

Molecular imaging-assisted optimization of hsp70 expression during laser-induced thermal preconditioning for wound repair enhancement.

Patients at risk for impaired healing may benefit from prophylactic measures aimed at improving wound repair. Several photonic devices claim to enhance repair by thermal and photochemical mechanisms. We hypothesized that laser-induced thermal preconditioning would enhance surgical wound healing that was correlated with hsp70 expression. Using a pulsed diode laser (lambda=1.85 microm, tau(p)=2 ms, 50 Hz, H=7.64 mJ cm(-2)), the skin of transgenic mice that contain an hsp70 promoter-driven luciferase was preconditioned 12 hours before surgical incisions were made. Laser protocols were optimized in vitro and in vivo using temperature, blood flow, and hsp70-mediated bioluminescence measurements as benchmarks. Biomechanical properties and histological parameters of wound healing were evaluated for up to 14 days. Bioluminescent imaging studies indicated that an optimized laser protocol increased hsp70 expression by 10-fold. Under these conditions, laser-preconditioned incisions were two times stronger than control wounds. Our data suggest that this molecular imaging approach provides a quantitative method for optimization of tissue preconditioning and that mild laser-induced heat shock may be a useful therapeutic intervention prior to surgery.

In-vivo optical imaging of hsp70 expression to assess collateral tissue damage associated with infrared laser ablation of skin.

Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 microm), protein (amide-II band, 6.45 microm), and both water and protein (amide-I band, 6.10 microm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 microm have approximately 4x higher hsp70 expression than 6.10 microm. Histology shows that under comparable fluences, tissue injury at the 2.94-microm wavelength was 2x and 3x deeper than 6.45 and 6.10 microm, respectively. The 6.10-microm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-microm wavelength is a superior wavelength for laser ablation of skin.

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair.

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

Regulation of the atheroma-enriched protein, SPRR3, in vascular smooth muscle cells through cyclic strain is dependent on integrin alpha1beta1/collagen interaction.

Atherosclerotic plaques express high levels of small proline-rich repeat protein (SPRR3), a previously characterized component of the cornified cell envelope of stratified epithelia, where it is believed to play a role in cellular adaptation to biomechanical stress. We investigated the physiological signals and underlying mechanism(s) that regulate atheroma-enriched SPRR3 expression in vascular smooth muscle cells (VSMCs). We showed that SPRR3 is expressed by VSMCs in both human and mouse atheromas. In cultured arterial VSMCs, mechanical cyclic strain, but neither shear stress nor lipid loading induced SPRR3 expression. Furthermore, this upregulation of SPRR3 expression was dependent on VSMC adherence to type I collagen. To link the mechanoregulation of SPRR3 to specific collagen/integrin interactions, we used blocking antibodies against either integrin alpha1 or alpha2 subunits and VSMCs from mice that lack specific collagen receptors. Our results showed a dependence on the alpha1beta1 integrin for SPRR3 expression induced by cyclic strain. Furthermore, we showed that integrin alpha1 but not alpha2 subunits were expressed on VSMCs within mouse lesions but not in normal arteries. Therefore, we identified the enrichment of the mechanical strain-regulated protein SPRR3 in VSMCs of both human and mouse atherosclerotic lesions whose expression is dependent on the collagen-binding integrin alpha1beta1 on VSMCs. These data suggest that SPRR3 may play a role in VSMC adaptation to local biomechanical stress within the plaque microenvironment.