Hanping Liu

Second-harmonic generation microscopy for assessment of mesenchymal stem cell-seeded acellular dermal matrix in wound-healing

Direct intra-skin injection of mesenchymal stem cells (MSCs) and the use of biomaterial scaffolds for grafts are both promising approaches of skin wound repair, however they still cannot generate skin that completely resembles the natural skin structures. In this study, we combined these two approaches by using acellular dermal matrix (ADM) recellularized with MSCs to repair cutaneous wounds in a murine model and two-photon fluorescence (TPF) microscopy and second-harmonic generation (SHG) microscopy to assess the effects of this therapy on wound healing. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were tagged with GFP and seeded into ADM (ADM-MSC) via MSC and ADM co-culture. ADM-MSC, ADM or saline was applied to murine excisional skin wounds and wound-healing was evaluated by histological examination on days 7, 14, 21 and TFP microscopy on days 1, 3, 5 and 21 post-treatment. ADM-MSC promoted healing significantly more than treatment with ADM or saline alone, as it led to substantial neovascularization and complete skin appendage regeneration. Furthermore, the SHG microscopic imaging technique proved to be a useful tool for monitoring changes in the collagen network at the wound site during the healing process and assessing the effects of different therapies.

Activities of MSCs Derived from Transgenic Mice Seeded on ADM Scaffolds in Wound Healing and Assessment by Advanced Optical Techniques

Background/Aims: Bone marrow Mesenchymal stem cells (MSCs) are promising for promoting cutaneous wound healing through reinforcing cellular processes. We evaluated the effect of GFP-tagged MSCs transplantation on skin regeneration in excisional wounds in mice. Methods: MSCs from GFP-labeled transgenic mice were co-cultured with acellular dermal matrix (ADM) scaffolds, and MSC-ADM scaffolds were transplanted into surgical skin wounds of BALB/c mice. After implantation, the survival and behavior of MSCs were examined by second harmonic generation and two-photon excitation fluorescence imaging, western blotting and DNA amplification and sequencing. Results: GFP-tagged MSCs were retained inside the regenerating skin until day 14 post-transplantation. Alpha-smooth muscle actin (α-SMA) and vimentin (VIM) were detected at 3, 5, 7, and 14 days post-transplantation by immunofluorescence double labeling. Although the GFP+/α-SMA+- and GFP+/VIM+-cell numbers decreased gradually with healing time, α-SMA+- and VIM+-cell numbers significantly increased, most of them were endogenous functional cells which were related to angiogenesis and collagen fiber structural remodeling. Conclusion: Therefore, in the initial stage of wound healing, transplanted MSCs differentiated into functional cells and played paracrine roles to recruit more endogenous cells for tissue remodeling. With the disappearance of exogenous cells, endogenous cells were responsible for the latter stage of cutaneous wound healing.

Label-free nonenzymatic glycation monitoring of collagen scaffolds in type 2 diabetic mice by confocal Raman microspectroscopy

Abstract. Collagen is the key target of nonenzymatic glycation during physiopathological processes such as diabetes. The induced changes in the biochemical property of collagen by nonenzymatic glycation remain a major challenge to probe. This study investigated the use of confocal Raman microspectroscopy to label-free monitor the nonenzymatic glycation of collagen scaffolds from type 2 diabetic (T2D) mice at different timepoints (0, 4, 8, and 12 weeks). The glycated collagen scaffolds were obtained through the decellularized dermal matrix method to remove the epidermis layer, subcutaneous tissue, and cells in the dermis and to retain the collagen fibrils. Raman spectra showed no changes in Raman peak positions, which indicated that nonenzymatic glycation could produce no significant changes in the triple-helix structure of collagen in T2D mice. However, the relative intensity of the Raman bands at 921, 1033, 1244, 1274, 1346, 1635, and 1672  cm−1 increased as diabetic time progressed. Correlation analysis suggested that the spectra of these bands had a high positive correlation with the expression of anti-advanced glycation end products obtained by immunofluorescence imaging of the same collagen scaffolds. Confocal Raman microspectroscopy proves a potential tool to label-free monitor the collagen changes caused by nonenzymatic glycation in T2D mice.

Backward emission angle of microscopic second-harmonic generation from crystallized type I collagen fiber

A theoretical model that deals with SHG from crystallized type I collagen fiber formed by a bundle of fibrils is established. By introducing a density distribution function of dipoles within fibrils assembly into the dipole theory and combining with structural order (m,l) parameters revealed by quasi-phase-matching (QPM) theory, our established theoretical model comprehensively characterizes both biophysical features of collagen dipoles and the crystalline characteristics of collagen fiber. This new model quantitatively reveals the 3-D distribution of second-harmonic generation (SHG) emission angle (θ,ϕ) in accordance with the emission power. Results show that fibrils diameter d(1) and structural order m, which describes the structural characteristics of collagen fiber along the incident light propagation direction has significant influence on backward∕forward SHG emission. The decrease of fibrils diameter d(1) induces an increase of the peak SHG emission angle θ(max). As d(1) decreases to a threshold value, in our case it is around d(1) = 150 nm when (m,l) = (1,0), θ(max) > 90 deg, indicating that backward SHG emission appears. The SHG may have two symmetrical emission distribution lobes or may have only one or two unsymmetrical emission lobes with unequal emission power, depending on the functional area of (m,l) on d(1).

Acellular dermal matrix from one-day-old mouse skin on adult scarless cutaneous wound repair by second harmonic generation microscopic imaging

In fetal skin, there is a process of scarless repair that does not exist in adults. The characteristics of the fetal skin extracellular matrix (ECM) are presumed to play a pivotal role for this scarless wound healing. The use of an acellular dermal matrix (ADM) with certain specific characteristics of the skin ECM thus is implied to have impact on skin repair and regeneration and the alteration of ADM properties may allow for the development of an effective intervention for scarless wound healing. In this study, two types of ADM from skin ECM were adopted to explore the potential to achieve scarless wound healing in adult mice. We transplanted this matrix onto the 10-week-old full-thickness cutaneous wound mice model. The ADM derived from the ECM of 1-day-old mouse skin (ADM-1D) was chosen to approximate certain characteristics of fetal skin while the ADM from the ECM of 20-week-old mouse skin (ADM-20W) provided a control with characteristics of mature skin. Second-harmonic generation (SHG) microscopic imaging was performed to dynamically demonstrate the collagen reconstruction process in the new born dermis during primary wound healing. The outcome of healing on day 21 was evaluated. Compared to ADM-20W, ADM-1D provided a more favorable influence on the re-establishment of the epidermis as well as collagen density, orientation and stiffness in the new born dermis, to a degree approaching normal uninjured adult dermal tissue. A remarkable difference in biomechanical stiffness is present between ADM-1D and ADM-20W, which might be one of the crucial determinants for potential adult scarless wound healing.

Simulation study of second-harmonic microscopic imaging signals through tissue-like turbid media.

We establish, for the first time, a simulation model for dealing with the second-harmonic signals under a microscope through a tissue-like turbid medium, based on the Monte Carlo method. With this model, the angle-resolved distribution and the signal level eta of second-harmonic light through a slab of the turbid medium are demonstrated and the effects of the thickness (d) of the turbid medium, the numerical aperture (NA) of the objective as well as the size (rho) of the scatterers forming the turbid medium are explored. Simulation results reveal that the use of a small objective NA results in a narrow angle distribution but strong second-harmonic signals. A turbid medium consisting of large scattering particles has a strong influence on the angle distribution and the signal level eta, which results in a low penetration limit for second-harmonic signals made up of ballistic photons. It is approximately 30 microm in our situation.

Curcumin-mediated bone marrow mesenchymal stem cell sheets create a favorable immune microenvironment for adult full-thickness cutaneous wound healing

BackgroundAdult full-thickness cutaneous wound repair suffers from an imbalanced immune response, leading to nonfunctional reconstructed tissue and fibrosis. Although various treatments have been reported, the immune-mediated tissue regeneration driven by biomaterial offers an attractive regenerative strategy for damaged tissue repair.MethodsIn this research, we investigated a specific bone marrow-derived mesenchymal stem cell (BMSC) sheet that was induced by the Traditional Chinese Medicine curcumin (CS-C) and its immunomodulatory effects on wound repair. Comparisons were made with the BMSC sheet induced without curcumin (CS-N) and control (saline).ResultsIn vitro cultured BMSC sheets (CS-C) showed that curcumin promoted the proliferation of BMSCs and modified the features of produced extracellular matrix (ECM) secreted by BMSCs, especially the contents of ECM structural proteins such as fibronectin (FN) and collagen I and III, as well as the ratio of collagen III/I. Two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging of mouse implantation revealed superior engraftment of BMSCs, maintained for 35 days in the CS-C group. Most importantly, CS-C created a favorable immune microenvironment. The chemokine stromal cell-derived factor 1 (SDF1) was abundantly produced by CS-C, thus facilitating a mass migration of leukocytes from which significantly increased expression of signature TH1 cells (interferon gamma) and M1 macrophages (tumor necrosis factor alpha) genes were confirmed at 7 days post-operation. The number of TH1 cells and associated pro-inflammatory M1 macrophages subsequently decreased sharply after 14 days post-operation, suggesting a rapid type I immune regression. Furthermore, the CS-C group showed an increased trend towards M2 macrophage polarization in the early phase. CS-C led to an epidermal thickness and collagen deposition that was closer to that of normal skin.ConclusionsCurcumin has a good regulatory effect on BMSCs and this promising CS-C biomaterial creates a pro-regenerative immune microenvironment for cutaneous wound healing.

MSCs on an acellular dermal matrix (ADM) sourced from neonatal mouse skin regulate collagen reconstruction of granulation tissue during adult cutaneous wound healing

Full-thickness cutaneous wound repair in adults usually leads to non-regenerative healing, which produces disorganized and non-functional fibrotic tissues. Mesenchymal stem cells (MSCs) on acellular dermal matrix (ADM) scaffolds are currently believed to be a promising strategy for wound healing improvement. Extracellular matrix (ECM) is aware of playing a pivotal role in wound healing, and changing the properties of the ECM is therefore expected to alter repair outcomes. Inspired by the analogous regeneration process in foetal skin with minimal or no scar tissue formation, in this study, ADM scaffold sourced from 1 day old mouse skin is introduced. Its influence on collagen reconstruction of granulation tissue during adult full-thickness wound healing is revealed and is compared to that from 20 week old mouse skin. Advanced nonlinear optical two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging were used to intravitally and three-dimensionally monitor the activities of MSCs and formation of granulation tissue. Dynamic changes of collagen content in granulation tissue were analyzed from aspects of synthesis and degradation. The associated collagen proteins, type I and type III collagen (Col-I and Col-III) and matrix matalloproteinase-13 (Mmp-13) were assessed at the transcriptional, translational and deposition levels. The results showed that there were significant differences in remodelling characteristics as a consequence of ADM properties. One-day old ADM + MSC treatment induced up-regulation of Col-III expression, down-regulation of Col-I and also down-regulation of Mmp-13. Accordingly, 1 day ADM + MSC treatment caused higher type III collagen deposition and a higher ratio of Col-III/Col-I in the granulation tissue. ADM derived from 1 day old skin ECM was superior to ADM derived from 20 week old skin ECM, caused enhanced angiogenesis, down regulation of TGF-β1 and promoted re-epithelization and faster, more constructive, and compositionally appropriate formation of granulation tissue. These results suggest that ADM derived from 1 day old skin ECM is a favourable biomaterial for adult wound healing.

Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signaling pathway

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin‐induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha‐smooth muscle actin and transforming growth factor‐beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing.

CXCR4 antagonist Delivery on Decellularized skin scaffold facilitates impaired wound healing in diabetic mice by increasing expression of SDF-1 and enhancing migration of CXCR4-positive cells

C‐X‐C chemokine receptor type 4 (CXCR4) is an alpha‐chemokine receptor specific for stromal cell‐derived factor 1 (SDF‐1 also called CXCL12). The antagonist of CXCR4 can mobilize CD34+ cells and hematopoietic stem cells from bone marrow within several hours, and it has an efficacy on diabetes ulcer through acting on the SDF‐1/CXCR4 axis. In this study, we investigated for the first time whether the antagonist of CXCR4 (Plerixafor/AMD3100) delivered on acellular dermal matrix (ADM) may accelerate diabetes‐impaired wound healing. ADM scaffolds were fabricated from nondiabetic mouse skin through decellularization processing and incorporated with AMD3100 to construct ADM‐AMD3100 scaffold. Full‐thickness cutaneous wound in streptozotocin (STZ)‐induced diabetic mice were treated with ADM, AMD3100, or ADM‐AMD3100. 21 days after treatment, wound closure in ADM‐AMD3100‐treated mice was more complete than ADM group and AMD3100 group, and it was accompanied by thicker collagen formation. Correspondingly, diabetic mice treated with ADM‐AMD3100 demonstrated prominent neovascularization (higher capillary density and vascular smooth muscle actin), which were accompanied by up‐regulated mRNA levels of SDF‐1 and enhanced migration of CXCR4 in the granulation tissue. Our results demonstrate that ADM scaffold provide perfect niche for loading AMD3100 and ADM‐AMD3100 is a promising method for diabetic wound healing mainly by increasing expression of SDF‐1 and enhancing migration of CXCR4‐positive cells.