Christopher S. Crowe

Optimization of an injectable tendon hydrogel: the effects of platelet-rich plasma and adipose-derived stem cells on tendon healing in vivo.

INTRODUCTION Acute and chronic tendon injuries would benefit from stronger and more expeditious healing. We hypothesize that supplementation of a biocompatible tendon hydrogel with platelet-rich plasma (PRP) and adipose-derived stem cells (ASCs) would augment the tendon healing process. MATERIALS AND METHODS Using 55 Wistar rats, a full-thickness defect was created within the midsubstance of each Achilles tendon with the addition of one of five experimental conditions: (i) saline control (50-μL), (ii) tendon hydrogel (50-μL), (iii) tendon hydrogel (45-μL)+PRP (5-μL), (iv) tendon hydrogel (45-μL)+2×10(6)-ASCs/mL in phosphate buffered saline (5-μL), and (v) tendon hydrogel (45-μL)+2×10(6)-ASCs/mL in PRP (5-μL). Hydrogel was developed from decellularized, human cadaveric tendons. Fresh rat PRP was obtained per Amable et al.s technique, and green fluorescent protein/luciferase-positive rat ASCs were utilized. Rats were sacrificed at weeks 1, 2, 4, and 8 after injury. Real-time in vivo bioluminescence imaging of groups with ASCs was performed. Upon sacrifice, Achilles tendons underwent biomechanical and histological evaluation. Comparisons across groups were analyzed using the two-sample Z-test for proportions and the Students t-test for independent samples. Significance was set at p<0.05. RESULTS (i) Bioluminescence imaging demonstrated that total photon flux was significantly increased for hydrogel+PRP+ASCs, versus hydrogel+ASCs for each postoperative day imaged (p<0.03). (ii) Mean ultimate failure load (UFL) was increased for hydrogel augmented with PRP and/or ASCs versus hydrogel alone at week 2 (p<0.03). By week 4, hydrogel alone reached a similar mean UFL to hydrogel augmented with PRP and/or ASCs (p>0.3). However, at week 8, hydrogel with PRP and ASCs demonstrated increased strength over other groups (p<0.05), except for hydrogel with PRP (p=0.25). (iii) Upon histological analysis, Hematoxylin and Eosin staining showed increased extracellular matrix formation in groups containing PRP and increased cellularity in groups containing ASCs. Groups containing both PRP and ASCs demonstrated both of these characteristics. CONCLUSION PRP and ASCs are easily accessible bioactive products that have potentiating effects on tendon hydrogel. Augmentation with these two factors encourages earlier mechanical strength and functional restoration. Thus, biochemically, tendon hydrogel augmented with PRP and/or ASCs, serves as a promising therapeutic modality for augmenting the tendon healing process after injury.

Tendon regeneration with a novel tendon hydrogel: in vitro effects of platelet-rich plasma on rat adipose-derived stem cells.

Background: Tendon hydrogel is a promising new injectable substance that has been shown to improve repair strength after tendon injury. This study assesses the capacity of platelet-rich plasma to stimulate proliferation and migration of rat adipose-derived stem cells in tendon hydrogel in vitro. Methods: To assess proliferation, adipose-derived stem cells were exposed to plasma, plasma supplemented with growth factors, or platelet-rich plasma in culture medium and tendon hydrogel. To assess migration, adipose-derived stem cells were plated onto tendon hydrogel -coated wells and covered with medium containing plasma, plasma supplemented with growth factors, platelet-rich plasma, or bovine serum albumin. Migration from cell-seeded to cell-free zones was assessed at 12-hour intervals. Results: Platelet-rich plasma augmented proliferation to a greater extent compared with plasma and plasma supplemented with growth factors (10%: optical density, 1.18 versus 0.75 versus 0.98, respectively). Platelet-rich plasma was superior to plasma in tendon hydrogel (10%: optical density, 1.19 versus 0.85) but did not augment proliferation to the extent that plasma supplemented with growth factors did (10%: optical density, 1.19 versus 1.56). Platelet-rich plasma enhanced the migration of adipose-derived stem cells compared with serum-free medium (bovine serum albumin) (36 hours: platelet-rich plasma, 1.88; plasma, 1.51; plasma plus growth factor, 1.80; bovine serum albumin, 1.43). Conclusions: Tendon healing is mediated by migration of cells to the injured area and cellular proliferation at that site. Tendon hydrogel supplemented with platelet-rich plasma stimulates these processes. Future studies will evaluate this combination’s ability to stimulate healing in chronic tendon injuries in vivo.

Tissue engineering in flexor tendon surgery: current state and future advances.

Tissue engineering of flexor tendons addresses a challenge often faced by hand surgeons: the restoration of function and improvement of healing with a limited supply of donor tendons. Creating an engineered tendon construct is dependent upon understanding the normal healing mechanisms of the tendon and tendon sheath. The production of a tendon construct includes: creating a three-dimensional scaffold; seeding cells within the scaffold; encouraging cellular growth within the scaffold while maintaining a gliding surface; and finally ensuring mechanical strength. An effective construct incorporates these factors in its design, with the ultimate goal of creating tendon substitutes that are readily available to the reconstructive hand surgeon.

Characteristics of Reconstituted Lyophilized Tendon Hydrogel: An Injectable Scaffold for Tendon Regeneration

Background: The authors have developed a tendon hydrogel that may be injected into the site of tendon injury to improve speed and strength of repair. The aim of this study was to compare the biological and physical properties of fresh, hydrated tendon hydrogel with its reconstituted lyophilized counterpart with the goal of increasing clinical feasibility. Materials: Hydrogel was prepared from fresh human cadaveric flexor tendon. Fresh gel was compared to gel aliquots that were lyophilized and reconstituted with sterile deionized water. Scanning electron microscopy was used to examine the microarchitecture of gelated samples. Rat adipose-derived stem cells were seeded in hydrogel, and cell viability was assessed after 7 days. MTS colorimetric assay was used to evaluate both the effect of prolonged storage on gel and the ability of reconstituted lyophilized hydrogel to activate platelet-rich plasma. The viability and proliferation of luciferase-transfected adipose-derived stem cells embedded within hydrogel in vivo was assessed by a bioluminescence in vivo imaging system. Results: Reconstituted lyophilized hydrogel demonstrated similar handling properties compared to fresh gel. Adipose-derived stem cells remained viable 7 days after reseeding in both conditions. Lyophilized hydrogel retained its ability to activate platelet-rich plasma and retained 95 percent of its maximal proliferative capacity at 30 days. The in vivo imaging system demonstrated similar cell proliferation, with signal persisting through day 13. Conclusions: Reconstitution of lyophilized hydrogel stimulated cell proliferation and platelet-rich plasma activation to a greater degree than did fresh hydrogel. Efficacy after prolonged storage was also shown to be superior. Therefore, this lyophilized formulation of tendon hydrogel may have wider clinical applicability.

In Vitro Characteristics of Porcine Tendon Hydrogel for Tendon Regeneration.

PurposePrevious work has characterized the development of a human tendon hydrogel capable of improving mechanical strength after tendon injury. Animal tendon hydrogel has not yet been described, but would prove beneficial due to the cost and ethical concerns associated with the use of human cadaveric tendon. This study details the manufacture and assesses the biocompatibility of porcine tendon hydrogel seeded with human adipoderived stem cells (ASCs). Materials and MethodsPorcine tendon was dissected from surrounding connective and muscle tissue and decellularized via 0.2% sodium dodecyl sulfate and 0.2% sodium dodecyl sulfate/ethylenediaminetetraacetic acid wash solutions before lyophilization. Tendon was milled and reconstituted by previously described methods. Decellularization was confirmed by hematoxylin-eosin staining, SYTO Green 11 nucleic acid dye, and DNeasy assay. The protein composition of milled tendon matrix before and after digestion was identified by mass spectrometry. Rheological properties were determined using an ARG2 rheometer. Biocompatibility was assessed by live/dead assay. The proliferation of human ASCs seeded in porcine and human hydrogel was measured by MTS assay. All experimental conditions were performed in triplicate. ResultsDecellularization of porcine tendon was successful. Mass spectrometry showed that collagen composes one third of milled porcine tendon before and after pepsin digestion. Rheology demonstrated that porcine hydrogel maintains a fluid consistency over a range of temperatures, unlike human hydrogel, which tends to solidify. Live/dead staining revealed that human ASCs survive in hydrogel 7 days after seeding and retain spindle-like morphology. MTS assay at day 3 and day 5 showed that human ASC proliferation was marginally greater in human hydrogel. ConclusionsAfter reconstitution and digestion, porcine hydrogel was capable of supporting growth of human ASCs. The minimal difference in proliferative capacity suggests that porcine tendon hydrogel may be an effective and viable alternative to human hydrogel for the enhancement of tendon healing.

The Tissue-engineered Tendon-bone Interface: In Vitro and In Vivo Synergistic Effects of Adipose-derived Stem Cells, Platelet-rich Plasma, and Extracellular Matrix Hydrogel

Background: Suboptimal healing of the tendon-bone interface remains an unsolved problem. The authors hypothesized that (1) platelet-rich plasma and prolonged in vitro incubation will produce interface scaffolds with greater reseeding of viable adipose-derived stem cells; and (2) when implanted with extracellular matrix hydrogel, constructs will display superior in vivo strength repair and biocompatibility. Methods: Achilles-calcaneal composite tendon-bone interface scaffold grafts were harvested from 30 Wistar rats. After physicochemical decellularization and lyophilization, scaffolds were revitalized in rat plasma or 100% activated rat platelet-rich plasma and reseeded with viable adipose-derived stem cells. For part 2 of the study, 90 Sprague-Dawley rats underwent reconstruction with one of five decellularized, lyophilized scaffold revitalization/reseeding conditions: (1) phosphate-buffered saline; (2) lyophilized, 100% activated platelet-rich plasma; (3) platelet-rich plasma and extracellular matrix hydrogel; (4) platelet-rich plasma and 14-day reseeding with ASC-luc2-eGFP cells; and (5) plasma, reseeding, and hydrogel. Results: In part 1, platelet-rich plasma–revitalized grafts demonstrated greater live viable adipose-derived stem cell loads at 3, 7, and 14 days and total adipose-derived stem cell loads at 7 and 14 days with visibly greater live surface cellularity, layering, migration, and penetration. In part 2, bioluminescence imaging confirmed cell viability to day 22 after implantation. Biomechanical strength testing demonstrated a significant increase in ultimate failure load for reseeded groups compared with all other groups at week 2, whereas only reseeded grafts with hydrogel remained significantly stronger at weeks 4 and 8. Histologic examination demonstrated most increased tendinous cellular invasion and fibrocartilage repopulation at 8 weeks in the reseeded group with hydrogel. Masson trichrome staining demonstrated persistence of the scaffold structure at week 8 and blinded ImageJ analysis demonstrated significantly more type III collagen in the reseeded/hydrogel group at 2, 4, and 8 weeks. Conclusions: Decellularized lyophilized allogeneic tendon-bone interface scaffolds can be optimized by revitalization in platelet-rich plasma, reseeding with viable adipose-derived stem cells, and supplemented by an extracellular matrix tendon hydrogel at the time of implantation. When this is done, they display greater repair strength and biocompatibility.

Beyond Phonosurgery: Considerations for Patient-Reported Outcomes and Speech Therapy in Transgender Vocal Feminization.

Providing appropriate care for transgender women is essential as more seek gender confirmation treatment for gender dysphoria. A variety of nonsurgical and surgical interventions are available. In terms of voice, pitch elevation in transgender women can have a significant effect on gender confirmation. Both surgical and nonsurgical options for pitch elevation exist. Phonosurgery can be an effective treatment; however, it is not without risk, and alternative nonsurgical interventions like voice therapy should be considered. A recent meta-analysis by Song and Jiang confirmed that transgender phonosurgery can result in significantly increased fundamental frequency (F0). However, lacking from this study was a mention of speech therapy’s role and effectiveness. Primary literature suggests F0 can be increased after speech therapy in transgender women, but no review of these data has been compiled. With a minimal risk profile, speech therapy has an important role in vocal feminization. Cost of surgery and limited availability of surgical specialists may be other reasons to consider speech therapy. Patient centeredness is paramount in treatment decision making, as it is the patients’ perspective and happiness with their voice that is the ultimate arbiter of success, not solely the ability to raise F0 with either voice therapy or surgery. We argue that the primary outcome for pitch elevation interventions should be patient centered. Interventions to change physiologic aspects of the voice should be judged based on whether the patient achieves improved quality of life and sense of self. Ultimately, it is the patient’s goals and perceptions that are most important. Successful outcomes in gender-confirming surgery are largely defined based on patients’ ability to live and be perceived as their true gender in all aspects of life. To date, little data exist on patient-reported outcomes after gender-confirming surgery, and when evaluated, the data are often nonstandardized and analyzed unreliably. Song and Jiang acknowledge this limitation in outcome assessment in primary literature. A standardized and reliable assessment of patient-reported outcomes after pitch elevation could add substantially to our understanding of the outcomes of pitch elevation. Transgender patients are often discriminated against and marginalized in the health care field. As the benefits of gender-confirming therapies are becoming more established, it is imperative that our surgical research be rigorous, standardized, and balanced, recognizing that nonsurgical interventions like voice therapy should also be strongly considered and studied. While we strive to deliver improved surgical interventions to better the quality-oflife outcomes to our transgender patients, we must not overlook effective nonsurgical options.

Tendon Regeneration with Tendon Hydrogel-Based Cell Delivery: A Comparison of Fibroblasts and Adipose-Derived Stem Cells

Background: Tendon hydrogel is a promising biomaterial for improving repair strength after tendon injury. This study compares the capacity of fibroblasts and adipose-derived stem cells to proliferate, survive, and acquire tenogenic properties when seeded into tendon hydrogel in vitro and in vivo. Methods: The effect of cell density on hydrogel contraction was measured macroscopically. To assess tenogenic properties, RNA was isolated from cells seeded in vitro in hydrogel, and tenocyte markers were quantified. To assess in vitro proliferation and survival, MTS and live-dead assays were performed. Finally, to assess the in vivo survival of cells in hydrogel, subcutaneous injections were performed on rats and in vivo imaging was performed. Results: At 0.5 million cells/ml, both the fibroblasts and adipose-derived stem cells induced minimal hydrogel contraction compared with higher cellular concentrations. Fibroblasts and adipose-derived stem cells seeded at 0.5 million cells/ml in tendon hydrogel up-regulated several tenocyte markers after 1 week. On MTS assay, fibroblasts and adipose-derived stem cells proliferated in hydrogel at similar rates. On live-dead assay, fibroblasts survived longer than adipose-derived stem cells. With use of the in vivo imaging system and histologic evaluation, fibroblasts survived longer than adipose-derived stem cells in hydrogel in vivo. Conclusions: Tendon healing is mediated by the proliferation, survival, and tenogenic differentiation of cells at the site of injury. Tendon hydrogel delivering dermal fibroblasts may improve and stimulate this process compared with adipose-derived stem cells. Future studies will be needed to evaluate the effects of this hydrogel-based cell delivery on chronic tendon injuries.

Hospital Transfer of Open Tibial Fractures Requiring Microsurgical Reconstruction Negatively Impacts Clinical Outcomes.

Introduction Open tibial fractures are orthopedic emergencies that may present with severe soft tissue damage. Free tissue transfer is often required when local tissues are insufficient and patients may require hospital transfer to tertiary care centers for this purpose. Although the negative impact of inter-facility transfer has been well demonstrated in trauma patients, less is known regarding transfers for lower extremity injury patients. This study investigates differences in outcomes based on transfer status after open tibial fracture. Methods Thirty-four consecutive open tibial fractures requiring free tissue transfer over a 13-year period (2001–2014) were retrospectively reviewed. Patients transferred from outside facilities were compared to non-transferred patients in regards to demographics, injury, surgical characteristics, and outcomes (mean follow-up of 2.5 years). Student t tests and chi square analysis were used to compare means and proportions, respectively. Results One-half of patients were transferred from an outside hospital. Transferred and nontransferred patients were not significantly different in regard to age, sex, comorbidities, mechanism of injury, and Gustilo grade. There was no significant difference in the method of bony fixation or free flap used for soft tissue coverage. The time to wound vacuum-assisted closure placement was longer in transferred patients, though this difference was not found to be significant (2.4 vs 3.3 days, P = 0.55). Time to definitive bony fixation was delayed in the transfer group by 9 days (5.2 vs 14.1 days, P = 0.05) and to tissue coverage by 7 days (14.2 vs 20.9 days, P = 0.13). Rates of flap loss and amputation did not differ between the groups. However, transferred patients were more likely to develop osteomyelitis (risk ratio [RR], 3.0; P = 0.03), nonunion (RR, 5.0; P = 0.09), and require hardware removal (RR, 3.3; P = 0.01). Conclusions Transferred and nontransferred patients were not significantly different in their demographics or presentation. However, an analysis of clinical outcomes showed that transfer was associated with increased rates of osteomyelitis, nonunion, and hardware removal. Although likely multifactorial, this study suggests that a delay to definitive fixation and soft tissue coverage contributes to the increased rate of complications and poorer prognosis.