Doctor of Philosophy, University of Virginia, Biomedical Engineering (2018)
Bachelor of Science, University of California, Berkeley, Bioengineering (2013)
Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/L, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.
View details for DOI 10.3390/mi11090815
View details for PubMedID 32872278
View details for Web of Science ID 000548418300023
Mechanical stimulation of the healing tendon is thought to regulate scar anisotropy and strength and is relatively easy to modulate through physical therapy. However, in vivo studies of various loading protocols in animal models have produced mixed results. To integrate and better understand the available data, we developed a multiscale model of rat Achilles tendon healing that incorporates the effect of changes in the mechanical environment on fibroblast behavior, collagen deposition, and scar formation. We modified an OpenSim model of the rat right hindlimb to estimate physiologic strains in the lateral/medial gastrocnemius and soleus musculo-tendon units during loading and unloading conditions. We used the tendon strains as inputs to a thermodynamic model of stress fiber dynamics that predicts fibroblast alignment, and to determine local collagen synthesis rates according to a response curve derived from in vitro studies. We then used an agent-based model (ABM) of scar formation to integrate these cell-level responses and predict tissue-level collagen alignment and content. We compared our model predictions to experimental data from ten different studies. We found that a single set of cellular response curves can explain features of observed tendon healing across a wide array of reported experiments in rats-including the paradoxical finding that repairing transected tendon reverses the effect of loading on alignment-without fitting model parameters to any data from those experiments. The key to these successful predictions was simulating the specific loading and surgical protocols to predict tissue-level strains, which then guided cellular behaviors according to response curves based on in vitro experiments. Our model results provide a potential explanation for the highly variable responses to mechanical loading reported in the tendon healing literature and may be useful in guiding the design of future experiments and interventions.
View details for DOI 10.1371/journal.pcbi.1006652
View details for Web of Science ID 000454835100044
View details for PubMedID 30550566
View details for PubMedCentralID PMC6310293
The ability of cells to orient in response to mechanical stimuli is essential to embryonic development, cell migration, mechanotransduction, and other critical physiologic functions in a range of organs. Endothelial cells, fibroblasts, mesenchymal stem cells, and osteoblasts all orient perpendicular to an applied cyclic stretch when plated on stretchable elastic substrates, suggesting a common underlying mechanism. However, many of these same cells orient parallel to stretch in vivo and in 3D culture, and a compelling explanation for the different orientation responses in 2D and 3D has remained elusive. Here, we conducted a series of experiments designed specifically to test the hypothesis that differences in strains transverse to the primary loading direction give rise to the different alignment patterns observed in 2D and 3D cyclic stretch experiments ("strain avoidance"). We found that, in static or low-frequency stretch conditions, cell alignment in fibroblast-populated collagen gels correlated with the presence or absence of a restraining boundary condition rather than with compaction strains. Cyclic stretch could induce perpendicular alignment in 3D culture but only at frequencies an order of magnitude greater than reported to induce perpendicular alignment in 2D. We modified a published model of stress fiber dynamics and were able to reproduce our experimental findings across all conditions tested as well as published data from 2D cyclic stretch experiments. These experimental and model results suggest an explanation for the apparently contradictory alignment responses of cells subjected to cyclic stretch on 2D membranes and in 3D gels.
View details for DOI 10.1073/pnas.1715059115
View details for Web of Science ID 000423728800055
View details for PubMedID 29343646
View details for PubMedCentralID PMC5798351
The purpose of this study was to determine the feasibility of using mouse models for translational study of flexor tendon repair and reconstruction.Quantitative data detailing the gross anatomy, biomechanical characteristics, and microscopic structure of the deep digit flexor tendon (DDF) of the mouse hindpaw were obtained. Histological characterization of the DDF and the anatomy of the digit in the mouse hindpaw are detailed. Biomechanical testing determined the load-to-failure, stress, elastic modulus, and the site of tendon failure.In gross anatomy, the origins and insertions of the mouse deep digit flexor tendon are similar to those of the human digit, surrounded by a synovial sheath that is only 1- to 2-cells thick. A neurovascular network runs on each side of the digit outside the synovial sheath, but does not clearly penetrate it. The thickness of the DDF is 0.14 ± 0.03?mm and the width is 0.3 ± 0.03?mm. The thickness of the DDF is less than that of 9-0 nylon needle. The mean failure force of the deep flexor tendon was 2.79 ± 0.53N.The gross anatomy of the mouse hindpaw digit is similar to that of the human digit except for key differences seen in the synovial sheath and vascular supply. The dimensions of the mouse DDF make it challenging to create a clinically translatable repair model using currently available surgical techniques. Despite the similarities between the human and mouse anatomy, and the powerful basic science tools available in murine models, mice are an unreliable model for assessing flexor tendon injury and repair.
View details for DOI 10.1097/GOX.0000000000003359
View details for PubMedID 33552814
View details for PubMedCentralID PMC7859083
Arteriovenous (AV) fistulas for hemodialysis can lead to cardiac volume loading and increased serum brain natriuretic peptide (BNP) levels. Whether short-term AV loop placement in patients undergoing microsurgery has an impact on cardiac biomarkers and circulating microRNAs (miRNAs), potentially indicating an increased hemodynamic risk, remains elusive. Fifteen patients underwent AV loop placement with delayed free flap anastomosis for microsurgical reconstructions of lower extremity soft-tissue defects. N-terminal pro-BNP (NT-proBNP), copeptin (CT-proAVP), and miRNA expression profiles were determined in the peripheral blood before and after AV loop placement. MiRNA expression in the blood was correlated with miRNA expression from AV loop vascular tissue. Serum NT-proBNP and copeptin levels exceeded the upper reference limit after AV loop placement, with an especially strong NT-proBNP increase in patients with preexistent cardiac diseases. A miRNA signature of 4 up-regulated (miR-3198, miR-3127-5p, miR-1305, miR-1288-3p) and 2 down-regulated miRNAs (miR30a-5p, miR-145-5p) which are related to cardiovascular physiology, showed a significant systemic deregulation in blood and venous tissue after AV loop placement. AV loop placement causes serum elevations of NT-proBNP, copeptin as well as specific circulating miRNAs, indicating a potentially increased hemodynamic risk for patients with cardiovascular comorbidities, if free flap anastomosis is delayed.
View details for DOI 10.1038/s41598-020-78905-y
View details for PubMedID 33311598
View details for Web of Science ID 000582792300339
View details for Web of Science ID 000582792300411
View details for Web of Science ID 000582798100088
View details for Web of Science ID 000582792300425
View details for Web of Science ID 000548418300026
View details for Web of Science ID 000548418300068
View details for Web of Science ID 000548418300050
View details for Web of Science ID 000548418300006
View details for Web of Science ID 000548418300144
Objective: To develop a novel approach for tissue engineering of soft-tissue flaps suitable for free microsurgical transfer, using an injectable nanofiber hydrogel composite (NHC) vascularized by an arteriovenous (AV) loop. Approach: A rat AV loop model was used for tissue engineering of vascularized soft-tissue flaps. NHC or collagen-elastin (CE) scaffolds were implanted into isolation chambers together with an AV loop and explanted after 15 days. Saphenous veins were implanted into the scaffolds as controls. Neoangiogenesis, ultrastructure, and protein expression of SYNJ2BP, EPHA2, and FOXC1 were analyzed by immunohistochemistry and compared between the groups. Rheological properties were compared between the two scaffolds and native human adipose tissue. Results: A functional neovascularization was evident in NHC flaps with its amount being comparable with CE flaps. Scanning electron microscopy revealed a strong mononuclear cell infiltration along the nanofibers in NHC flaps and a trend toward higher fiber alignment compared with CE flaps. SYNJ2BP and EPHA2 expression in endothelial cells (ECs) was lower in NHC flaps compared with CE flaps, whereas FOXC1 expression was increased in NHC flaps. Compared with the stiffer CE flaps, the NHC flaps showed similar rheological properties to native human adipose tissue. Innovation: This is the first study to demonstrate the feasibility of tissue engineering of soft-tissue flaps with similar rheological properties as human fat, suitable for microsurgical transfer using an injectable nanofiber hydrogel composite. Conclusions: The injectable NHC scaffold is suitable for tissue engineering of axially vascularized soft-tissue flaps with a solid neovascularization, strong cellular infiltration, and biomechanical properties similar to human fat. Our data indicate that SYNJ2BP, EPHA2, and FOXC1 are involved in AV loop-associated angiogenesis and that the scaffold material has an impact on protein expression in ECs.
View details for DOI 10.1089/wound.2019.0975
View details for PubMedID 32587789
View details for PubMedCentralID PMC7307685
Cryopreserved human skin allografts (CHSAs) are used for the coverage of major burns when donor sites for autografts are insufficiently available and have clinically shown beneficial effects on chronic non-healing wounds. However, the biologic mechanisms behind the regenerative properties of CHSA remain elusive. Furthermore, the impact of cryopreservation on the immunogenicity of CHSA has not been thoroughly investigated and raised concerns with regard to their clinical application. To investigate the importance and fate of living cells, we compared cryopreserved CHSA with human acellular dermal matrix (ADM) grafts in which living cells had been removed by chemical processing. Both grafts were subcutaneously implanted into C57BL/6 mice and explanted after 1, 3, 7, and 28?days (n = 5 per group). A sham surgery where no graft was implanted served as a control. Transmission electron microscopy (TEM) and flow cytometry were used to characterise the ultrastructure and cells within CHSA before implantation. Immunofluorescent staining of tissue sections was used to determine the immune reaction against the implanted grafts, the rate of apoptotic cells, and vascularisation as well as collagen content of the overlaying murine dermis. Digital quantification of collagen fibre alignment on tissue sections was used to quantify the degree of fibrosis within the murine dermis. A substantial population of live human cells with intact organelles was identified in CHSA prior to implantation. Subcutaneous pockets with implanted xenografts or ADMs healed without clinically apparent rejection and with a similar cellular immune response. CHSA implantation largely preserved the cellularity of the overlying murine dermis, whereas ADM was associated with a significantly higher rate of cellular apoptosis, identified by cleaved caspase-3 staining, and a stronger dendritic cell infiltration of the murine dermis. CHSA was found to induce a local angiogenic response, leading to significantly more vascularisation of the murine dermis compared with ADM and sham surgery on day 7. By day 28, aggregate collagen-1 content within the murine dermis was greater following CHSA implantation compared with ADM. Collagen fibre alignment of the murine dermis, correlating with the degree of fibrosis, was significantly greater in the ADM group, whereas CHSA maintained the characteristic basket weave pattern of the native murine dermis. Our data indicate that CHSAs promote angiogenesis and collagen-1 production without eliciting a significant fibrotic response in a xenograft model. These findings may provide insight into the beneficial effects clinically observed after treatment of chronic wounds and burns with CHSA.
View details for DOI 10.1111/iwj.13349
View details for PubMedID 32227459
Intravenous infusion of mesenchymal stromal cells (MSCs) is thought to be a viable treatment for numerous disorders. Although the intrinsic immunosuppressive ability of MSCs has been credited for this therapeutic effect, their exact impact on endogenous tissue-resident cells following delivery has not been clearly characterized. Moreover, multiple studies have reported pulmonary sequestration of MSCs upon intravenous delivery. Despite substantial efforts to improve MSC homing, it remains unclear whether MSC migration to the site of injury is necessary to achieve a therapeutic effect. Using a murine excisional wound healing model, we offer an explanation of how sequestered MSCs improve healing through their systemic impact on macrophage subpopulations. We demonstrate that infusion of MSCs leads to pulmonary entrapment followed by rapid clearance, but also significantly accelerates wound closure. Using single-cell RNA sequencing of the wound, we show that following MSC delivery, innate immune cells, particularly macrophages, exhibit distinctive transcriptional changes. We identify the appearance of a pro-angiogenic CD9+ macrophage subpopulation, whose induction is mediated by several proteins secreted by MSCs, including COL6A1, PRG4, and TGFB3. Our findings suggest that MSCs do not need to act locally to induce broad changes in the immune system and ultimately treat disease.
View details for DOI 10.1016/j.ymthe.2020.05.022
View details for PubMedID 32531238
Burn injury in the craniofacial region causes significant health and psychosocial consequences and presents unique reconstructive challenges. Healing of severely burned skin and underlying soft tissue is a dynamic process involving many pathophysiological factors, often leading to devastating outcomes such as the formation of hypertrophic scars and debilitating contractures. There are limited treatment options currently used for post-burn scar mitigation but recent advances in our knowledge of the cellular and molecular wound and scar pathophysiology have allowed for development of new treatment concepts. Clinical effectiveness of these experimental therapies is currently being evaluated. In this review, we discuss current topical therapies for craniofacial burn injuries and emerging new therapeutic concepts that are highly translational.
View details for DOI 10.3389/fphys.2020.00916
View details for PubMedID 32848859
View details for PubMedCentralID PMC7403506