Ulnar Wrist Denervation: Articular Branching Pattern and Selective Blockade of the Dorsal Branch of the Ulnar Nerve.

PURPOSE: Ulnar wrist denervation has been a successful treatment for patients with ulnar-sided wrist pain. The purpose of this study was to characterize the articular branches of the dorsal branch of the ulnar nerve (DBUN) and validate a technique for selective peripheral nerve blockade. METHODS: In cadavers, we performed simulated local anesthetic injections using 0.5 mL of 0.5% methylene into the subcutaneous tissue at a point midway between the palpable borders of the pisiform and ulnar styloid. We then dissected the DBUN, characterized its articular branching pattern, and measured staining intensity of the DBUN and the ulnar nerve relative to a standard. RESULTS: The DBUN branched from the ulnar nerve 7.0 ± 1.2 cm proximal to the ulnar styloid. Among 17 specimens, the DBUN provided an average of 1.2 (range, 0-2) ulnocarpal branches and 1.0 (range, 0-2) carpometacarpal articular branches. A simulated local anesthetic injection successfully stained 100% of the DBUN articular branches at or proximal to their takeoff. There was no staining of the proper ulnar nerves. In all specimens, the DBUN supplied at least one articular branch. CONCLUSIONS: A point midway between the palpable border of the pisiform and ulnar styloid may be an effective location for selectively blocking the DBUN articular afferents. CLINICAL RELEVANCE: In this study, we were able to identify a point halfway between the pisiform and ulnar styloid that has the potential to produce a selective peripheral nerve block of the portion of the DBUN that supplies articular fibers to the ulnocarpal joint and the fifth carpometacarpal joint. This technique may prove useful to surgeons treating ulnar-sided wrist pain.

Targeted muscle reinnervation prevents and reverses rat pain behaviors after nerve transection.

ABSTRACT: Targeted muscle reinnervation (TMR) is a clinical intervention that is rapidly becoming common in major limb amputation to prevent or reduce amputation-related pain. However, TMR is much less effective when applied long after injury compared with acute TMR. Since the mechanisms governing pain relief in TMR of amputated nerves are unknown, we developed a preclinical model as a platform for mechanistic examination. Following spared nerve injury (SNI), rats underwent either TMR, simple neuroma excision, or a sham manipulation of the injury site. These interventions were performed immediately or delayed (3 or 12 weeks) after SNI. Pain behavior was measured as sensitivity to mechanical stimuli (pin, von Frey, and dynamic brush) and thermal stimuli (acetone and radiant heat). Spared nerve injury produced hypersensitivity to all mechanical stimuli and cold, which persisted after sham surgery. Targeted muscle reinnervation at the time of SNI prevented the development of pain behaviors and performing TMR 3 weeks after SNI reversed pain behaviors to baseline. By contrast, TMR performed at 12 weeks after SNI had no effect on pain behaviors. Neuroma excision resulted in significantly less reduction in hyperalgesia compared with TMR when performed 3 weeks after SNI but had no effect at 12 weeks after SNI. In this model, the pain phenotype induced by nerve transection is reduced by TMR when performed within 3 weeks after injury. However, TMR delayed 12 weeks after injury fails to reduce pain behaviors. This replicates clinical experience with limb amputation, supporting validity of this model for examining the mechanisms of TMR analgesia.

Postoperative Interventions in Pediatric Digital Replantation: A Tertiary Referral Center Case Review.

PURPOSE: Although a few case series have been published describing the excellent outcomes of replantation and revascularization operations in children, there has been limited study of the hospital course that these patients experience and the number of potentially harmful interventions and treatments that occur. The purpose of this study was to detail the results of various postoperative interventions, including anticoagulation, transfusion, leeching, sedation, and additional anesthetic exposures. METHODS: Twenty-nine patients aged less than 18 years had 34 digital revascularizations or replantations performed between January 2000 and May 2020. The details of each patient's presentation, surgery, and postoperative care were analyzed. RESULTS: Nine of 29 children underwent repeat anesthetics, including 6 revision amputations. No demographic, surgical, or postoperative variables consistently preceded revision amputation or additional anesthetic procedures. Only 5 patients had >1 hemoglobin (Hb) measurement. Two patients received blood transfusions; the average drop in Hb was 3.5 g/dL from before surgery to the lowest after surgery. Four patients underwent leech therapy. Only patients receiving leech therapy required postoperative transfusions. Anticoagulation regimens were prescribed on the basis of demographic and surgical factors, although no medication or regimen seemed to affect outcomes. CONCLUSIONS: Although the experience of digital replantation is essentially the same in pediatric patients as adults, there may be different ramifications for children. Specifically, postoperative management of pediatric digital replantation or revascularization can involve multiple interventions that carry their risks. Parents should be counseled about the risks of anticoagulants, transfusions, and repeat anesthetics, and clinicians should monitor Hb closely when using leech therapy. TYPE OF STUDY/LEVEL OF EVIDENCE: Case series, Therapeutic IV.

Challenges and Potential in Targeted Muscle Reinnervation in Pediatric Amputees.

Targeted muscle reinnervation (TMR) is a powerful new tool in preventing and treating residual limb and phantom limb pain. In the adult population, TMR is rapidly becoming standard of care; however, there is a paucity of literature regarding indications and outcomes of TMR in the pediatric population. We present 2 cases of pediatric patients who sustained amputations and the relevant challenges associated with TMR in their cases. One is a 7-year-old patient who developed severe phantom and residual limb pain after a posttraumatic above-knee amputation. He failed pharmacologic measures and underwent TMR. He obtained complete relief of his symptoms and is continuing to do well 1.5 years postoperatively. The other is a 2-year-old boy with bilateral wrist and below-knee amputations as sequelae of sepsis. TMR was not performed because the patient never demonstrated evidence of phantom limb pain or symptomatic neuroma formation. We use these 2 cases to explore the challenges particular to pediatric patients when considering treatment with TMR, including capacity to report pain, risks of anesthesia, and cortical plasticity. These issues will be critical in determining how TMR will be applied to pediatric patients.

Human cells and networks of pain: Transforming pain target identification and therapeutic development.

Chronic pain is a disabling disease with limited treatment options. While animal models have revealed important aspects of pain neurobiology, therapeutic translation of this knowledge requires our understanding of these cells and networks of pain in humans. We propose a multi-institutional collaboration to rigorously and ethically address this challenge.

Early Experience With Locked Intramedullary Wrist Arthrodesis.

PURPOSE: The most common method of total wrist arthrodesis is dorsal compression plating, which can require revision for soft tissue or tendon irritation. A locked intramedullary system was developed to reduce this complication. The goal of this study was to investigate the complication rate of total wrist arthrodesis using this system in our center. METHODS: A retrospective chart review of all patients undergoing intramedullary wrist arthrodesis from January 2016 to February 2018 was performed. RESULTS: Nine wrist arthrodeses were performed with locked intramedullary wrist fusion in 5 women and 4 men. The indications for fusion included posttraumatic arthritis in 7 and inflammatory arthritis in 2. Two patients underwent primary arthrodesis and 7 had revision from prior partial wrist fusions. Local bone graft at the radiocarpal joint was used in all cases. The mean follow-up was 27 weeks. Radiocarpal fusion was achieved in 8 patients after the initial procedure and in the ninth patient after revision. There were 6 complications in 4 patients with revision reoperation required in 3. The complications were metacarpal locking screw migration in 3, metacarpal fracture in 1, radiocarpal nonunion in 1, and symptomatic middle finger carpometacarpal nonunion in 1. The revision surgeries include distal screw removal in 1, distal screw removal with replacement in 1, and bone grafting in the radiocarpal nonunion. CONCLUSIONS: Use of the locked intramedullary wrist fusion system yields high fusion rates. However, based on the high complication rate, particularly from distal screw migration, and the high revision rate in this series, we recommend caution with the use of this system. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

Nerve transfer surgery in cervical spinal cord injury: a qualitative study exploring surgical and caregiver participant experiences.

PURPOSE: To investigate perceptions of surgical participants and their caregivers regarding novel nerve transfer surgery to restore upper extremity function in cervical level spinal cord injury. MATERIALS AND METHODS: A qualitative study design was used. A multidisciplinary team developed semi-structured interview guides. Interviews were recorded, transcribed and analyzed using basic text analysis. RESULTS: Participants had limited information about procedures to improve function after spinal cord injury. When discussing their choice to undergo nerve (as compared to traditional tendon) transfer surgery, they describe a desire to avoid post-operative immobilization. Barriers included the pre-operative testing, cost and inconvenience of travel for surgery, and understanding complex health information related to the procedure. While expectations matched descriptions of outcomes among participants and were generally positive, caregivers expressed disappointment. The long time interval for gains in function to be realized and relatively incremental gains achieved were frustrating to all. CONCLUSIONS: People with cervical spinal cord injury and their caregivers need more information about options to restore function and about realistic range of improvements with treatment. Further work to mitigate barriers and develop health information materials around nerve transfer surgery may improve medical decision making around and appropriate use of this newer treatment option.IMPLICATIONS FOR REHABILITATIONNerve transfer surgery is a novel and acceptable means of improving upper extremity function in the setting of cervical spinal cord injury.People with cervical spinal cord injury and their caregivers need information about options to restore hand and arm function and mitigation of barriers around these treatment options.

Successful Digital Revascularization in a Patient With Factor V Leiden Mutation.

Replantation/revascularization involves microsurgical anastomosis of the digital arteries and veins, which are often 2 mm or less in diameter. Thrombosis is a known risk of revascularization that ultimately can lead to ischemic digital loss. Factor V Leiden mutation is present in 3% to 8% of the population and results in generalized hypercoagulability. We present the case of a single-digit revascularization that was successful following vein grafting in a patient with factor V Leiden mutation.

Increasing Nerve Autograft Length Increases Senescence and Reduces Regeneration.

BACKGROUND: Nerve grafting with an autograft is considered the gold standard. However, the functional outcomes of long (>3 cm) nerve autografting are often poor. The authors hypothesized that a factor contributing to these outcomes is the graft microenvironment, where long compared to short autografts support axon regeneration to different extents. METHODS: A rat sciatic nerve defect model was used to compare regeneration in short (2 cm) and long (6 cm) isografts. Axon regeneration and cell populations within grafts were assessed using histology, retrograde labeling of neurons regenerating axons, immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction, and electron microscopy at 4 and/or 8 weeks. RESULTS: At 8 weeks, for distances of both 1 and 2 cm from the proximal coaptation (equivalent regenerative distance), long isografts had reduced numbers of regenerated fibers compared with short isografts. Similarly, the number of motoneurons regenerating axons was reduced in the presence of long isografts compared with short isografts. Considering the regenerative microenvironments between short and long isografts, cell densities and general populations within both short and long isografts were similar. However, long isografts had significantly greater expression of senescence markers, which included senescence-associated ß-galactosidase, p21, and p16, and distinct chromatin changes within Schwann cells. CONCLUSIONS: This study shows that axon regeneration is reduced in long compared with short isografts, where long isografts contained an environment with an increased accumulation of senescent markers. Although autografts are considered the gold standard for grafting, these results demonstrate that we must continue to strive for improvements in the autograft regenerative environment.

The Use of Nerve Transfers to Restore Upper Extremity Function in Cervical Spinal Cord Injury.

BACKGROUND: Nerve transfer surgery to restore upper extremity function in cervical spinal cord injury (SCI) is novel and may transform treatment. Determining candidacy even years post-SCI is ill defined and deserves investigation. OBJECTIVE: To develop a diagnostic algorithm, focusing on electrodiagnostic (EDX) studies, to determine eligibility for nerve transfer surgery. DESIGN: Retrospective descriptive case series. SETTING: Tertiary university-based institution. PATIENTS: Individuals with cervical SCI (n = 45). METHODS: The electronic medical records of people referred to the Plastic Surgery Multidisciplinary Upper Extremity Surgery in SCI clinic from 2010-2015 were reviewed. People were considered for nerve transfers to restore elbow extension or finger flexion and/or extension. Data including demographic, clinical evaluation, EDX results, surgery, and outcomes were collected and analyzed. MAIN OUTCOME MEASUREMENTS: EDX data, including nerve conduction studies and electromyography, for bilateral upper extremities of each patient examined was used to assess for the presence of lower motor neuron injury, which would preclude late nerve transfer. RESULTS: Based on our criteria and the results of EDX testing, a substantial number of patients presenting even years post-SCI were candidates for nerve transfers. Clinical outcome results are heterogeneous but promising and suggest that further refinement of eligibility, long-term follow-up, and standardized assessment will improve our understanding of the role of nerve transfer surgery to restore function in people with midcervical SCI. CONCLUSIONS: Many patients living with SCI are candidates for nerve transfer surgery to restore upper extremity function. Although the ultimate efficacy of these surgeries is not yet determined, this study attempts to report the criteria we are using and may ultimately determine the timing for intervention and which transfers are most useful for this heterogeneous population. LEVEL OF EVIDENCE: IV.

Endoscope-assisted management of sagittal synostosis: wide vertex suturectomy and barrel stave osteotomies versus narrow vertex suturectomy.

OBJECTIVE Endoscope-assisted methods for treatment of craniosynostosis have reported benefits over open calvarial vault reconstruction. In this paper, the authors evaluated 2 methods for endoscope-assisted correction of sagittal synostosis: wide vertex suturectomy and barrel stave osteotomies (WVS+BSO) and narrow vertex suturectomy (NVS). METHODS The authors evaluated patients with nonsyndromic sagittal synostosis treated with either wide vertex suturectomy (4-6 cm) and barrel stave osteotomies (WVS+BSO) or narrow vertex suturectomy (NVS) (approximately 2 cm) between October 2006 and July 2013. Prospectively collected data included patient age, sex, operative time, estimated blood loss (EBL), postoperative hemoglobin level, number of transfusions, complications, and cephalic index. Fourteen patients in the NVS group were age matched to 14 patients in the WVS+BSO group. Descriptive statistics were calculated, and Student t-tests were used to compare prospectively obtained data from the WVS+BSO group with the NVS group in a series of univariate analyses. RESULTS The mean age at surgery was 3.9 months for WVS+BSO and 3.8 months for NVS. The mean operative time for patients undergoing NVS was 59.0 minutes, significantly less than the 83.4-minute operative time for patients undergoing WVS+BSO (p < 0.05). The differences in mean EBL (NVS: 25.4 ml; WVS+BSO: 27.5 ml), mean postoperative hemoglobin level (NVS: 8.6 g/dl; WVS+BSO: 8.0 g/dl), mean preoperative cephalic index (NVS: 69.9; WVS+BSO: 68.2), and mean cephalic index at 1 year of age (NVS: 78.1; WVS+BSO: 77.2) were not statistically significant. CONCLUSIONS The NVS and WVS+BSO produced nearly identical clinical results, as cephalic index at 1 year of age was similar between the 2 approaches. However, the NVS required fewer procedural steps and significantly less operative time than the WVS+BSO. The NVS group obtained the final cephalic index in a similar amount of time postoperatively as the WVS+BSO group. Complications, transfusion rates, and EBL were not different between the 2 techniques.

Axonal Growth Arrests After an Increased Accumulation of Schwann Cells Expressing Senescence Markers and Stromal Cells in Acellular Nerve Allografts.

Acellular nerve allografts (ANAs) and other nerve constructs do not reliably facilitate axonal regeneration across long defects (>3 cm). Causes for this deficiency are poorly understood. In this study, we determined what cells are present within ANAs before axonal growth arrest in nerve constructs and if these cells express markers of cellular stress and senescence. Using the Thy1-GFP rat and serial imaging, we identified the time and location of axonal growth arrest in long (6 cm) ANAs. Axonal growth halted within long ANAs by 4 weeks, while axons successfully regenerated across short (3 cm) ANAs. Cellular populations and markers of senescence were determined using immunohistochemistry, histology, and senescence-associated ß-galactosidase staining. Both short and long ANAs were robustly repopulated with Schwann cells (SCs) and stromal cells by 2 weeks. Schwann cells (S100ß(+)) represented the majority of cells repopulating both ANAs. Overall, both ANAs demonstrated similar cellular populations with the exception of increased stromal cells (fibronectin(+)/S100ß(-)/CD68(-) cells) in long ANAs. Characterization of ANAs for markers of cellular senescence revealed that long ANAs accumulated much greater levels of senescence markers and a greater percentage of Schwann cells expressing the senescence marker p16 compared to short ANAs. To establish the impact of the long ANA environment on axonal regeneration, short ANAs (2 cm) that would normally support axonal regeneration were generated from long ANAs near the time of axonal growth arrest ("stressed" ANAs). These stressed ANAs contained mainly S100ß(+)/p16(+) cells and markedly reduced axonal regeneration. In additional experiments, removal of the distal portion (4 cm) of long ANAs near the time of axonal growth arrest and replacement with long isografts (4 cm) rescued axonal regeneration across the defect. Neuronal culture derived from nerve following axonal growth arrest in long ANAs revealed no deficits in axonal extension. Overall, this evidence demonstrates that long ANAs are repopulated with increased p16(+) Schwann cells and stromal cells compared to short ANAs, suggesting a role for these cells in poor axonal regeneration across nerve constructs.

Vascularization is delayed in long nerve constructs compared with nerve grafts.

INTRODUCTION: Nerve regeneration across nerve constructs, such as acellular nerve allografts (ANAs), is inferior to nerve auto/isografts especially in the case of long defect lengths. Vascularization may contribute to poor regeneration. The time course of vascular perfusion within long grafts and constructs was tracked to determine vascularization. METHODS: Male Lewis rat sciatic nerves were transected and repaired with 6 cm isografts or ANAs. At variable days following grafting, animals were perfused with Evans Blue albumin, and grafts were evaluated for vascular perfusion by a blinded observer. RESULTS: Vascularization at mid-graft was re-established within 3-4 days in 6 cm isografts, while it was established after 10 days in 6 cm ANAs. CONCLUSIONS: Vascular perfusion is reestablished over a shorter time course in long isografts when compared with long ANAs. The differences in vascularization of long ANAs compared with auto/isografts suggest regenerative outcomes across ANAs could be affected by vascularization rates. Muscle Nerve 54: 319-321, 2016.

Nerve Transfers to Restore Upper Extremity Function in Cervical Spinal Cord Injury: Update and Preliminary Outcomes.

BACKGROUND: Cervical spinal cord injury can result in profound loss of upper extremity function. Recent interest in the use of nerve transfers to restore volitional control is an exciting development in the care of these complex patients. In this article, the authors review preliminary results of nerve transfers in spinal cord injury. METHODS: Review of the literature and the authors' cases series of 13 operations in nine spinal cord injury nerve transfer recipients was performed. Representative cases were reviewed to explore critical concepts and preliminary outcomes. RESULTS: The nerve transfers used expendable donors (e.g., teres minor, deltoid, supinator, and brachialis) innervated above the level of the spinal cord injury to restore volitional control of missing function such as elbow extension, wrist extension, and/or hand function (posterior interosseous nerve or anterior interosseous nerve/finger flexors reinnervated). Results from the literature and the authors' patients (after a mean postsurgical follow-up of 12 months) indicate gains in function as assessed by both manual muscle testing and patients' self-reported outcomes measures. CONCLUSIONS: Nerve transfers can provide an alternative and consistent means of reestablishing volitional control of upper extremity function in people with cervical level spinal cord injury. Early outcomes provide evidence of substantial improvements in self-reported function despite relatively subtle objective gains in isolated muscle strength. Further work to investigate the optimal timing and combination of nerve transfer operations, the combination of these with traditional treatments (tendon transfer and functional electrical stimulation), and measurement of outcomes is imperative for determining the precise role of these operations. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Comparison of acellular nerve allograft modification with Schwann cells or VEGF.

BACKGROUND: Individual contributions of exogenous Schwann cells (SCs) and vascular endothelial growth factor (VEGF) were evaluated in acellular nerve allografts (ANAs). ANA processing removes SCs and vasculature, likely contributing to reduced regeneration compared to autografts. Exogenous SCs may improve the regenerative microenvironment, and VEGF has been shown to stimulate angiogenesis. Replacing these components in ANAs may improve regeneration. METHODS: A rat sciatic nerve transection model was used to study 20-mm grafts. Four graft types were studied: (1) isograft, (2) ANA, (3) ANA-SCs, and (4) ANA-VEGF. After 10 weeks in vivo, the midgraft and distal nerve to the grafts were analyzed for axonal regeneration using histomorphometry to assess total myelinated axon counts, density, width, and percent neural tissue. RESULTS: The most axons in the distal nerve were regenerated in the isograft followed by the ANA- SC group, with 9171 ± 1822 and 7103 ± 1576 regenerated axons respectively. Both the ANA and ANA-VEGF groups had significantly fewer regenerated axons compared to the isograft (p < 0.05) with 5225 ± 2994 and 5709 ± 2657 regenerated axons, respectively. The ANA and ANA-VEGF groups also had significantly reduced fiber density and percent nerve compared to the isograft; the isograft and ANA-SC groups were not significantly different (p < 0.05). CONCLUSIONS: These results show that SCs improve axonal regeneration in a 20 mm ANA to a greater extent than VEGF. VEGF treatment showed a trend toward increased axonal regeneration but was not significantly different compared to the untreated ANA. The role of VEGF may be clearer in longer grafts where ischemia is a greater factor.

Use of peripheral nerve transfers in tetraplegia: evaluation of feasibility and morbidity.

BACKGROUND: Peripheral nerve transfers are being used to improve upper extremity function in cervical spinal cord injury (SCI) patients. The purpose of this study was to evaluate feasibility and perioperative complications following these procedures. METHODS: Eligible SCI patients with upper extremity dysfunction were assessed and followed for a minimum of 3 months after surgery. Data regarding demographics, medical history, physical examination, electrodiagnostic testing, intraoperative nerve stimulation, recipient nerve histomorphometry, surgical procedure, and complications were collected. RESULTS: Seven patients had surgery on eight limbs, mean age of 28 ± 9.9 years and mean time from SCI injury of 5.1 ± 5.2 years. All patients had volitional elbow flexion and no volitional hand function. The nerve to the brachialis muscle was used as the expendable donor, and the recipients included the anterior interosseous nerve (AIN) (for volitional prehension), nerve branches to the flexor carpi radialis, and flexor digitorum superficialis. Two patients underwent additional nerve transfers: (1) supinator to extensor carpi ulnaris or (2) deltoid to triceps. No patients had any loss of baseline upper extremity function, seven of eight AIN nerve specimens had preserved micro-architecture, and all intraoperative stimulation of recipient neuromuscular units was successful further supporting feasibility. Four patients had perioperative complications; all resolved or improved (paresthesias). CONCLUSION: Nerve transfers can be used to reestablish volitional control of hand function in SCI. This surgery does not downgrade existing function, uses expendable donor nerve, and has no postoperative immobilization, which might make it a more viable option than traditional tendon transfer and other procedures.

Tissue augmentation with fibrin sealant and cultured fibroblasts: a preliminary study.

BACKGROUND: Nonoperative subdermal tissue augmentation is one of the most frequently performed procedures in plastic surgery and dermatological practice. Many products, from biological to synthetic filler substances, are currently available. However, none has achieved ideal clinical efficacy, especially regarding volume maintenance and longevity. We examined the use of fibrin sealant as a biological and fully degradable matrix for dermal augmentation in combination with precultured human fibroblasts and hyaluronic acid gel (HYAFF). METHODS: Four implant preparations were studied: fibrin glue only (F); 1% HYAFF mixed in fibrin glue (FH); 1.8 × 10(6) cells/ml of fibrin glue (FC); and 1% HYAFF and 1.8 × 10(6) cells/ml of fibrin glue (FHC). Each mouse was given two separate subcutaneous injections of implant material. At 1, 3, and 6 weeks two mice from each group were sacrificed, such that there was an n = 4 for each implant group at each time point. The mice were grossly examined for implant retention and the implants were evaluated by means of immunohistochemistry for fibrosis, integration into surrounding tissue, presence of elastin, and blood vessel infiltration. RESULTS: Only the implants in the cell-containing groups, FC and FHC, remained after 6 weeks. Moreover, with the exception of a mild inflammatory response, no adverse affects of the cell-seeded implants were noted. CONCLUSION: Presence of fibroblasts increases implant durability. Further studies should evaluate the ideal hyaluronic acid and fibroblast concentration for long-term longevity.

Fibrochondrogenesis of hESCs: growth factor combinations and cocultures.

The successful differentiation of human embryonic stem cells (hESCs) to fibrochondrocyte-like cells and characterization of these differentiated cells is a critical step toward tissue engineering of musculoskeletal fibrocartilages (e.g., knee meniscus, temporomandibular joint disc, and intervertebral disc). In this study, growth factors and primary cell cocultures were applied to hESC embryoid bodies (EBs) for 3 weeks and evaluated for their effect on the synthesis of critical fibrocartilage matrix components: glycosaminoglycans (GAG) and collagens (types I, II, and VI). Changes in surface markers (CD105, CD44, SSEA, PDGFR alpha) after the differentiation treatments were also analyzed. The study was conducted in three phases: (1) examination of growth factors (TGF-beta 3, BMP-2, BMP-4, BMP-6, PDGF-BB, sonic hedgehog protein); (2) comparison of two cocultures (primary chondrocytes or fibrochondrocytes); and (3) the combination of the most effective growth factor and coculture regimen. TGF-beta 3 with BMP-4 yielded EBs positive for collagens I, II, and VI, with up to 6.7- and 4.8-fold increases in GAG and collagen, respectively. Analysis of cell surface markers showed a significant increase in CD44 with the TGF-beta 3 + BMP-4 treatment compared to the controls. Coculture with fibrochondrocytes resulted in up to a 9.8-fold increase in collagen II production. The combination of the growth factors BMP-4 + TGF-beta 3 with the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. This study determined two powerful treatments for inducing fibrocartilaginous differentiation of hESCs and provides a foundation for using flow cytometry to purify these differentiated cells.

Use of staurosporine, an actin-modifying agent, to enhance fibrochondrocyte matrix gene expression and synthesis.

Modulation of the actin cytoskeleton in chondrocytes has been used to prevent or reverse dedifferentiation and to enhance protein synthesis. We have hypothesized that an actin-modifying agent, staurosporine, could be used with fibrochondrocytes to increase the gene expression and synthesis of critical fibrocartilage proteins. A range of concentrations (0.1-100 nM) was applied to fibrochondrocytes in monolayer and evaluated after 24 h and after 4 days. High-dose staurosporine treatment (10-100 nM) increased cartilage oligomeric matrix protein 60- to 500-fold and aggrecan gene expression two-fold. This effective range of staurosporine was then applied to scaffoldless tissue-engineered fibrochondrocyte constructs for 4 weeks. Whereas glycosaminoglycan synthesis was not affected, collagen content doubled, from 27.6 +/- 8.8 microg in the untreated constructs to 55.2 +/- 12.2 microg per construct with 100 nM treatment. When analyzed for specific collagens, the 10-nM group showed a significant increase in collagen type I content, whereas collagen type II was unaffected. A concomitant dose-dependent reduction was noted in construct contraction, reflecting the actin-disrupting action of staurosporine. Thus, staurosporine increases the gene expression for important matrix proteins and can be used to enhance matrix production and reduce contraction in tissue-engineered fibrocartilage constructs.