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.

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.

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.

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.

Creating a spectrum of fibrocartilages through different cell sources and biochemical stimuli.

In this study a scaffoldless approach was employed with two different cell sources and biochemical stimuli to engineer a spectrum of fibrocartilages representative of the different regions of the knee meniscus. Constructs composed of 100% fibrochondrocytes (FC) or a 50:50 co-culture of fibrochondrocytes and chondrocytes (CC) were cultured in 10% fetal bovine serum medium or serum-free "chondrogenic" medium, each +/-10 ng/mL TGF-beta1 (+T). Constructs from these two cell groups and four culture conditions were cultured for 6 weeks. By varying the cell type and presence of the growth factor, GAG per dry weight of the constructs spanned that of native tissue, ranging 16-45% and 1-7% in the CC and FC groups, respectively. Collagen density was most dependent on cell type and was significantly lower than tissue values. The collagen I/II ratio could be manipulated by cell type and serum presence to span the native range, from 3.5 in the serum-free CC group to over 1,000 in the FC groups treated with serum-containing medium. Using the CC cell group in the presence of serum-free medium dramatically increased the compressive stiffness to 128 +/- 34 kPa, similar to native tissue. Similarly, serum-free medium or TGF-beta1 treatment enhanced the tensile modulus by an order of magnitude, up to 3,000 kPa. Using two cell sources and manipulating biochemical stimuli, a range of fibrocartilaginous neotissues was engineered. Fibrocartilages such as the knee meniscus are characterized by heterogeneity in matrix and functional properties, and this work demonstrates a strategy for recreating these heterogeneous tissues.

Fibrochondrogenesis in two embryonic stem cell lines: effects of differentiation timelines.

Human embryonic stem cells (hESCs) are an exciting cell source for fibrocartilage engineering. In this study, the effects of differentiation time and cell line, H9 versus BG01V, were examined. Embryoid bodies (EBs) were fibrochondrogenically differentiated for 1, 3, or 6 weeks and then used to engineer tissue constructs that were grown for an additional 4 weeks. Construct matrix was fibrocartilaginous, containing glycosaminoglycans (GAGs) and collagens I, II, and VI. A differentiation time of 3 or 6 weeks produced homogeneous constructs, with matrix composition varying greatly with cell line and differentiation time: from 2.6 to 17.4 microg of GAG per 10(6) cells and from 22.3 to 238.4 microg of collagen per 10(6) cells. Differentiation for 1 week resulted in small constructs with poor structural integrity that could not be mechanically tested. The compressive stiffness of the constructs obtained from EBs differentiated for 3 or 6 weeks did not vary significantly as a function of either differentiation time or cell line. In contrast, the tensile properties were markedly greater with the H9 cell line, 1,562-1,940 versus 32-80 kPa in the BG01V constructs. These results demonstrate the dramatic effects of hESC line and differentiation time on the biochemical and functional properties of tissue-engineered constructs and show progress in fibrocartilage tissue engineering with an exciting new cell source.

Self-assembly of fibrochondrocytes and chondrocytes for tissue engineering of the knee meniscus.

Chondrocyte self-assembly in high-density scaffoldless culture has shown success in producing articular cartilage constructs, and a similar process could be applied to fibrocartilage tissue engineering. Three cell combinations were compared in self-assembly culture-100% chondrocytes, 100% meniscal fibrochondrocytes, and 50:50 co-cultures of fibrochondrocytes and chondrocytes with the goal of creating a proteoglycan, collagen I, and collagen II matrix similar to native meniscus. Two culture surfaces were also compared for self-assembly: agarose-coated wells and tissue culture plastic. After 4 weeks, the resulting self-assembled chondrocyte constructs were 10.24+/-0.63 mm in diameter and 0.96+/-0.14 mm thick, weighing 84.5+/-7.2 mg. Co-culture constructs were smaller and weighed 22.5+/-1.0 mg. In contrast, the fibrochondrocyte constructs contracted into spheres weighing 1.3+/-0.3 mg. Immunostaining showed collagen II in the chondrocyte constructs, both collagen I and collagen II in the co-cultures, and only collagen I in the fibrochondrocyte constructs. Collagen densities for chondrocyte, co-culture, and fibrochondrocyte constructs were 41+/-3, 38+/-3, and 20+/-2 microg/mg dry weight, and glycosaminoglycan densities were 230+/-2, 80+/-6, and 10+/-1 microg/mg dry weight, respectively. Self-assembled co-cultures, with their mixed collagen I and II matrix and robust gross characteristics, appear promising for tissue engineering of the knee meniscus.

Meniscal repair with fibrocartilage engineering.

Injuries to the knee meniscus, particularly those in the avascular region, pose a complex problem and a possible solution is tissue engineering of a replacement tissue. Tissue engineering of the meniscus involves scaffold selection, addition of cells, and stimulation of the construct to synthesize, maintain, or enhance matrix production. An acellular collagen implant is currently in clinical trials and there are promising results with other scaffolds, composed of both polymeric and natural materials. The addition of cells to these constructs may promote good matrix production in vitro, but has been studied in a limited manner in animal studies. Cell sources ranging from fibroblasts to stem cells could be used to overcome challenges in cell procurement, expansion, and synthetic capacity currently encountered in studies with fibrochondrocytes. Manipulation of construct culture with exogenous growth factors and mechanical stimulation will also likely play a role in these strategies.