Grace Chiou

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.

Comparing Muscle and Fasciocutaneous Free Flaps in Lower Extremity Reconstruction--Does It Matter?

IntroductionPlastic surgeons are often asked to assist with the reconstruction of lower extremity wounds. These patients many times require free tissue transfer for coverage given paucity of soft tissue. Anecdotally, many orthopedic surgeons prefer muscle coverage—particularly in the setting of potentially infected bone. Todays surgeons now easily harvest and transfer fasciocutaneous flaps—a versatile option with less donor-site morbidity. We hypothesized that there would be no difference in outcomes between these 2 types of reconstruction. MethodsWe performed a single-institution retrospective review of lower extremity free flap reconstructions in the last 10 years. Demographics, preoperative and postoperative course, and the documented time to weight-bearing and bony union were collected. Major cohorts compared were muscle free flaps and fasciocutaneous free flaps, further divided into subgroups including acute trauma, tumor resection, osteomyelitis, and nonunion. Data comparisons were made using paired t test and Fischer exact tests. ResultsThere were 121 patients who met inclusion criteria—86 in the muscle flap group, and 35 in the fasciocutaneous group and demographics were equal. Total complication rates were higher in smokers than nonsmokers (P < 0.03). There was no significant difference in major or minor complication rates between muscle and fasciocutaneous flaps in any subgroup. In both the acute fracture group and the infected nonunion group, there was a significantly faster return to weight bearing in the fasciocutaneous group (P < 0.03) although there was no difference in documented time to bony union. Patients who underwent fasciocutaneous reconstruction were more likely to require revisionary surgery for improved aesthetics (P < 0.001). ConclusionsOur data suggest that in essentially all clinical parameters, there is no difference between free flap type used for soft tissue coverage of the lower extremity. Patients undergoing reconstruction with a fasciocutaneous flap may return to weight bearing earlier—although they are more likely to require elective flap revisions. These results imply essentially equivalent outcomes regardless of flap type or operative indication, in contrast with some of the biases in the orthopedic community. The particular flap chosen for any reconstruction should remain solely at the discretion of the plastic 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.

Concurrent Laparoscopic Morgagni Hernia Repair and Sleeve Gastrectomy

The Morgagni-type anterior diaphragmatic hernia is a congenital defect that is a very uncommon hernia presenting in an adult. Surgical repair is usually recommended upon diagnosis and often requires synthetic mesh for a durable, tension-free repair. The use of synthetic mesh concurrently with several of bariatric operations is controversial owing to the potential for mesh infection. In this report we describe a laparoscopic repair of a symptomatic Morgagni hernia with synthetic mesh, concurrently with sleeve gastrectomy, in a morbidly obese man. The patient was a 58-year-old man with a body mass index of 48 kg/m(2) and associated co-morbid conditions that included obstructive sleep apnea, hypertension, hyperlipidemia, impaired fasting glucose, and osteoarthritis. He was diagnosed with Morgagni hernia with exertional dyspnia. He underwent concurrent laparoscopic Morgagni hernia repair with mesh and sleeve gastrectomy. At 2 months after surgery the patient was doing well and tolerating solid foods, and his percentage excess weight loss was 35%. He was exercising regularly and had no exertional dyspnea. Laparoscopy is an attractive approach to performing multiple intra-abdominal procedures concurrently. The Morgagni hernia repair with mesh can be performed safely and effectively using a laparoscopic approach. This can be performed concurrently with bariatric surgery in the morbidly obese.

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.

Refinements and Secondary Surgery After Flap Reconstruction of the Traumatized Hand

The traumatized hand often has soft tissue loss requiring flap reconstruction. Before proceeding with flap selection, the need for future refinement and secondary surgery should be taken into consideration. Although muscle flaps may offer better contour, fasciocutaneous flaps allow easier secondary flap elevation. After the initial flap reconstruction, indications for secondary procedures may be managed according to tissue type: bone, joint, tendon, nerve, and soft tissue.