Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2.

The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis.

NELL-1 based demineralized bone graft promotes rat spine fusion as compared to commercially available BMP-2 product.

BACKGROUND: Spinal fusion is among the most commonly performed orthopaedic procedures. Unfortunately, current treatments such as autologous bone grafting or recombinant proteins (BMP-2) have numerous clinical shortcomings. Here, we directly compare the efficacy of NELL-1, a novel osteoinductive growth factor, to two currently available treatments, (1) recombinant BMP-2 and (2) iliac crest bone grafting, in a spinal fusion model. METHODS: Twenty-six skeletally mature athymic rats underwent posterolateral spine fusion of L4/L5 vertebrae. Treatment groups included NELL-1 (10 and 50 µg) in a demineralized bone matrix (DBX), as compared to BMP-2 (90 µg) in an absorbable collagen sponge (ACS) or morselized iliac crest bone. Scaffolds without recombinant protein were used as controls. Animals were sacrificed at 4 weeks post-operative and fusion was assessed by manual palpation, radiography [high-resolution X-ray, micro-computed tomography (microCT)], histology (hematoxylin and eosin, Masson's trichrome) and immunohistochemistry (osteocalcin). RESULTS: Results showed 100 % fusion in all NELL-1- and BMP-2-treated samples. In contrast, lower rates of fusion were observed in scaffold-only and bone graft treatment groups. MicroCT scans revealed radiographic evidence of fusion among spines treated with NELL-1. Bone bridging was also observed with BMP-2 treatment, but was accompanied by inner radiolucency, suggesting cyst-like bone formation. Histologically, NELL-1-treated grafts showed increased bone formation, endochondral ossification and vascularization. Although BMP-2 treated grafts exhibited increased bone formation and angiogenesis, numerous adipocytes were also observed. CONCLUSION: NELL-1-based bone grafts are comparable to BMP-2 + ACS in spinal fusion efficacy. Histological differences were observed however, including robust endochondral ossification with NELL-1 treatment as compared to lipid-filled bone with BMP-2 treatment. These findings suggest NELL-1 based bone grafts show promise for future efforts in skeletal tissue engineering.

Epstein-Barr virus reactivation-related meningoencephalitis with transverse myelitis in pregnancy.

Key Clinical Message: Consider the differential of Epstein-Barr virus (EBV) reactivation in pregnant women who develop progressive meningoencephalitis and transverse myelitis. EBV nucleic acid amplification should be considered in immunosuppressed patients. Abstract: A 32-year-old G10P6M3K22 pregnant female presented to a regional hospital with progressive severe neurological and behavioral deficits. Magnetic resonance revealed cervical transverse myelitis. Lumbar puncture confirmed Epstein-Barr virus (EBV) DNA on a background of IgG-positive EBV serology. A diagnosis of EBV reactivation-related meningoencephalitis with transverse myelitis in pregnancy was concluded.

P-wave durations from automated electrocardiogram analysis to predict atrial fibrillation and mortality in heart failure.

BACKGROUND: P-wave indices have been used to predict incident atrial fibrillation (AF), stroke, and mortality. However, such indices derived from automated ECG measurements have not been explored for their predictive values in heart failure (HF). We investigated whether automated P-wave indices can predict adverse outcomes in HF. METHODS: This study included consecutive Chinese patients admitted to a single tertiary centre, presenting with HF but without prior AF, and with at least one baseline ECG, between 1 January 2010 and 31 December 2016, with last follow-up of 31 December 2019. RESULTS: A total of 2718 patients were included [median age: 77.4, interquartile range (IQR): (66.9-84.3) years; 47.9 males]. After a median follow-up of 4.8 years (IQR: 1.9-9.0 years), 1150 patients developed AF (8.8/year), 339 developed stroke (2.6/year), 563 developed cardiovascular mortality (4.3/year), and 1972 had all-cause mortality (15.1/year). Compared with 101-120 ms as a reference, maximum P-wave durations predicted new-onset AF at ≤90 ms [HR: 1.17(1.11, 1.50), P < 0.01], 131-140 ms [HR: 1.29(1.09, 1.54), P < 0.001], and ≥141 ms [HR: 1.52(1.32, 1.75), P < 0.001]. Similarly, they predicted cardiovascular mortality at ≤90 ms [HR: 1.50(1.08, 2.06), P < 0.001] or ≥141 ms [HR: 1.18(1.15, 1.45), P < 0.001], and all-cause mortality at ≤90 ms [HR: 1.26(1.04, 1.51), P < 0.001], 131-140 ms [HR: 1.15(1.01, 1.32), P < 0.01], and ≥141 ms [HR: 1.31(1.18, 1.46), P < 0.001]. These remained significant after adjusting for significant demographics, past co-morbidities, P-wave dispersion, and maximum P-wave amplitude. CONCLUSIONS: Extreme values of maximum P-wave durations (≤90 ms and ≥141 ms) were significant predictors of new-onset AF, cardiovascular mortality, and all-cause mortality.

Calvarial cleidocraniodysplasia-like defects with ENU-induced Nell-1 deficiency.

Nell-1, first identified by its overexpression in synostotic cranial sutures, is a novel osteoinductive growth and differentiation factor. To further define Nell-1's role in craniofacial patterning, we characterized defects of the ENU-induced Nell-1-deficient (END) mice, focusing on both intramembranous and endochondral cranial bones. Results showed that calvarial bones of neonatal END mice were reduced in thickness and density, with a phenotype resembling calvarial cleidocraniodysplasia. In addition, a global reduction in osteoblast markers was observed, including reductions in Runx2, alkaline phosphatase, and osteocalcin. Remarkably, detailed analysis of endochondral bones showed dysplasia as well. The chondrocranium in the END mouse showed enrichment for early, proliferating Sox9⁺ chondrocytes, whereas in contrast markers of chondrocytes maturation were reduced. These data suggest that Nell-1 is an important growth factor for regulation of osteochondral differentiation, by regulating both Runx2 and Sox9 expression within the calvarium. In summary, Nell-1 is required for normal craniofacial membranous and endochondral skeletal development.

Comparison of Dance-Related Foot and Ankle Injuries Among Pre-Professional Ballet, Contemporary, and Chinese Dancers.

BACKGROUND: Foot and ankle injuries have been noted as the most common in dancers. However, the variability of injury epidemiology across different dance genres has not been clearly evaluated. Herein, this study aimed to evaluate the prevalence and incidence of foot and ankle problems in pre-professional ballet, contemporary, and Chinese dancers.
Methods: Participants (N = 54) were recruited from a local dance institution that offered a formal undergraduate dance program. Demographic characteristics and specifics of foot and ankle pain during dancing were collected through an online self-reporting survey from September 2018 to June 2019. Descriptive statistical analyses, including injury incidence and risk rates, were conducted.
Results: The overall response rate was 69.3%, with a total of 88 subjects eligible for analysis of which the results from 54 subjects were ultimately analyzed. The incidence of foot and ankle pain during the academic year of 2018 to 2019 was highest in contemporary dancers (0.38 per 1,000 dance hours) when compared to that of ballet (0.32 per 1,000 dance hours) and Chinese dancers (0.22 per 1,000 dance hours). Prevalence of foot and ankle pain within the same year was 84% in ballet dancers, 79% in Chinese dancers, and 70% in contemporary dancers. Ballet dancers were six times more likely to suffer from pain in the Achilles region than Chinese and contemporary dancers (p < 0.01). Chinese dancers were found to experience more forefoot and midfoot problems compared to ballet and contemporary dancers (p < 0.05).
Conclusion: This study illustrated that foot and ankle pain is highly prevalent among pre-profes- sional dancers. Ballet was associated with the highest prevalence of foot-ankle pain while contemporary dance was associated with the highest incidence. There were significant differences of foot and ankle pain among dance genres and anatomical subregions, which suggests a need for targeted genre-specific injury prevention programs in hopes of preventing potentially career-ending injuries in dancers.

Avascular necrosis of the first metatarsal head in a young female adult: A case report and review of literature.

BACKGROUND: This case study describes an atypical presentation of avascular necrosis (AVN) of the first metatarsal head, which is largely unfounded in the literature. CASE SUMMARY: A healthy 24-year-old female initially presented with pain at the first metatarsophalangeal joint (MTPJ) and was diagnosed with AVN by physical examination and magnetic resonance imaging. The patient demonstrated atypically poor progress in recovery, despite being in otherwise good health and being of young age, with no history of corticosteroid or alcohol use. The patient also did not have any history or clinical features of autoimmune disease or vasculitis, such as systemic lupus erythematosus. The patient was managed with conservative treatment for 18 mo, which allowed for gradual return of full range of motion of the first MTPJ and subsiding pain, permitting the patient to return to high-intensity sports training and full weight-bearing. Throughout her recovery, many differential diagnoses were ruled out through specific investigations leading to further reinforcement of the diagnosis of AVN of the 1st metatarsal head. CONCLUSION: Atypical AVN may occur with no predisposing risk factors. Treatment is mainly conservative, with unclear guidelines in literature on management.

Prognosis of elite basketball players after an Achilles tendon rupture.

OBJECTIVE: Professional Basketball players are at high risk of Achilles tendon rupture. Despite this, there remains limited research into the factors affecting rehabilitation and the long term outcomes of these players. Our aim is to quantify the effect of a player's Achilles tendon rupture on their post-injury performance, and also to explore for correlations between their recovery timeline and pre-injury characteristics. Creation of an injury timeline of past incidents will allow injured players to better track their progress and also inform them about the probable impact on their careers. HYPOTHESIS: Players with Achilles tendon rupture injury will exhibit decreased performance compared to their pre-injury self and their non-injured peers after recovery. METHODS: Professional basketball players who sustained a unilateral Achilles tendon rupture from 1992 to 2016 were collected. 12 players met our inclusion criteria and their Player Efficiency Ratings (PER) were obtained as primary outcome measures; matched controls were chosen based on the PER, Age and playing position. The players' index season PER was compared against the PER during the 10 games immediately following the players' return and the PER of their post-injury peak performing season. The same data analysis was performed against their control group. To investigate the factors affecting the recovery and long-term consequences of their injury, we correlated the variables of Age, BMI, Time of Injury and pre-injury PER with the player's time to return to play and their post-injury PER. RESULTS: 2 out of 12 players failed to return to playing in the elite professional league following an Achilles Tendon Rupture, others returned after a mean recovery time of 10 months. When compared to players' index PER, the mean PER reduction during the 10 games immediately following the players' return was 7.15 (P < .000). Players on average took 1.8 seasons to reach their post-injury peak performance, with only 1 player returning to his pre-injury performance. Others suffered a mean PER reduction of 3.5 (P = .004) when compared to their index PER and 5.4 (P = .045) against their matched controls. CONCLUSION: Achilles tendon rupture can be a career-ending injury for professional basketball players. They are expected to miss 10 months for rehabilitation and reach their post-injury peak performance level at the 2nd season back. The post-injury peak performance is significantly worse than the pre-injury level, but is similar to matched non-injured players.

NELL-1 promotes cartilage regeneration in an in vivo rabbit model.

Repair of cartilage due to joint trauma remains challenging due to the poor healing capacity of cartilage and adverse effects related to current growth factor-based strategies. NELL-1 (Nel-like molecule-1; Nel [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]), a protein first characterized in the context of premature cranial suture fusion, is believed to accelerate differentiation along the osteochondral lineage. We previously demonstrated the ability of NELL-1 protein to maintain the cartilaginous phenotype of explanted rabbit chondrocytes in vitro. Our objective in the current study is to determine whether NELL-1 can affect endogenous chondrocytes in an in vivo cartilage defect model. To generate the implant, NELL-1 was incorporated into chitosan nanoparticles and embedded into alginate hydrogels. These implants were press fit into 3-mm circular osteochondral defects created in the femoral condylar cartilage of 3-month-old New Zealand White rabbits (n=10). Controls included unfilled defects (n=8) and defects filled with phosphate-buffered saline-loaded chitosan nanoparticles embedded in alginate hydrogels (n=8). Rabbits were sacrificed 3 months postimplantation for histological analysis. Defects filled with alginate containing NELL-1 demonstrated significantly improved cartilage regeneration. Remarkably, histology of NELL-1-treated defects closely resembled that of native cartilage, including stronger Alcian blue and Safranin-O staining and increased deposition of type II collagen and absence of the bone markers type I collagen and Runt-related transcription factor 2 (Runx2) as demonstrated by immunohistochemistry. Our results suggest that NELL-1 may produce functional cartilage with properties similar to native cartilage, and is an exciting candidate for tissue engineering-based approaches for treating diverse pathologies of cartilage defects and degeneration.

Use of human perivascular stem cells for bone regeneration.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized). FSDs are bicortical and are stabilized with a polyethylene bar and K-wires. The FSD described is also a critical size defect, which does not significantly heal on its own. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

The antimicrobial and osteoinductive properties of silver nanoparticle/poly (DL-lactic-co-glycolic acid)-coated stainless steel.

Implant-associated bacterial infections are one of the most serious complications in orthopedic surgery. Treatment of these infections often requires multiple operations, device removal, long-term systemic antibiotics, and extended rehabilitation, and is frequently ineffective, leading to worse clinical outcomes and increased financial costs. In this study, we evaluated silver nanoparticle/poly(DL-lactic-co-glycolic acid) (PLGA)-coated stainless steel alloy(SNPSA) as a potential antimicrobial implant material. We found that SNPSA exhibited strong antibacterial activity in vitro and ex vivo, and promoted MC3T3-E1 pre-osteoblasts proliferation and maturation in vitro. Furthermore, SNPSA implants induced osteogenesis while suppressing bacterial survival in contaminated rat femoral canals. Our results indicate that SNPSA has simultaneous antimicrobial and osteoinductive properties that make it a promising therapeutic material in orthopedic surgery.

Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells.

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.

Nfatc2 is a primary response gene of Nell-1 regulating chondrogenesis in ATDC5 cells.

Nell-1 is a growth factor required for normal skeletal development and expression of extracellular matrix proteins required for bone and cartilage cell differentiation. We identified the transcription factor nuclear factor of activated T cells (Nfatc2) as a primary response gene of Nell-1 through a microarray screen, with validation using real-time polymerase chain reaction (PCR). We investigated the effects of recombinant Nell-1 protein on the chondrogenic cell line ATDC5 and primary mouse chondrocytes. The osteochondral transcription factor Runx2 was investigated as a possible intermediary between Nell-1 and Nfatc2 using adenoviral overexpression of wild-type and dominant-negative Runx2. Nell-1 transiently induced both transcription and translation of Nfatc2, an effect inhibited by transduction of dominant-negative Runx2, suggesting that Runx2 was necessary for Nfatc2 induction. Differentiation assays revealed inhibitory effects of Nell-1 on ATDC5 cells. Although proliferation was unaffected, expression of chondrocyte-specific genes was decreased, and cartilage nodule formation and proteoglycan accumulation were suppressed. siRNA knockdown of Nfatc2 significantly reversed these inhibitory effects. To elucidate the relationship between Nell-1, Runx2, and Nfatc2 in vivo, their presence and distribution were visualized in femurs of wild-type and Nell1-deficient mice at both neonatal and various developmental stages using immunohistochemistry. All three proteins colocalized in the perichondrium of wild-type femurs but stained weakly or were completely absent in Nell1-deficient femurs at neonatal stages. Thus Nfatc2 likely plays an important role in Nell-1-mediated osteochondral differentiation in vitro and in vivo. To our knowledge, this is the first demonstration that Nfatc2 is a primary response gene of Nell-1.

Nell-1 enhances bone regeneration in a rat critical-sized femoral segmental defect model.

BACKGROUND: Effective regeneration of bone is critical for fracture repair and incorporation and healing of bone grafts used during orthopedic, dental, and craniofacial reconstructions. Nel-like molecule-1 (Nell-1) is a secreted protein identified from prematurely fused cranial sutures of craniosynostosis patients that has been found to specifically stimulate osteogenic cell differentiation and bone formation. To test the in vivo osteoinductive capacity of Nell-1, a critical-sized femoral segmental defect model in athymic rats was used. METHODS: A 6-mm defect, which predictably leads to nonunion if left untreated, was created in the left femur of each rat. Three treatment groups (n = 8 each) were created consisting of rats treated with (1) 1.5 mg/ml Nell-1, (2) 0.6 mg/ml Nell-1, and (3) phosphate-buffered saline only as a Nell-free control. Phosphate-buffered saline or Nell-1 was mixed with demineralized bone matrix as a carrier before implantation. All animals were euthanized 12 weeks after surgery, and bone regeneration was evaluated using radiographic, three-dimensional micro-computed tomographic, and histologic analysis. RESULTS: Both Nell-1-treated groups had significantly greater bone formation compared with the Nell-free group, with bone volume increasing with increasing Nell-1 concentration. CONCLUSIONS: Nell-1 in a demineralized bone matrix carrier can significantly improve bone regeneration in a critical-sized femoral segmental defect in a dose-dependent manner. The results of this study demonstrate that Nell-1 is a potent osteospecific growth factor that warrants further investigation. Results also support the potential application of Nell-1 as a bone graft substitute in multiple clinical scenarios involving repair of critical bone loss when autograft bone is limited or unavailable.

Nell-1 protein promotes bone formation in a sheep spinal fusion model.

Bone morphogenetic proteins (BMPs) are widely used as bone graft substitutes in spinal fusion, but are associated with numerous adverse effects. The growth factor Nel-like molecule-1 (Nell-1) is mechanistically distinct from BMPs and can minimize complications associated with BMP therapies. This study evaluates the efficacy of Nell-1 combined with demineralized bone matrix (DBM) as a novel bone graft material for interbody spine fusion using sheep, a phylogenetically advanced animal with biomechanical similarities to human spine. Nell-1+sheep DBM or Nell-1+heat-inactivated DBM (inDBM) (to determine the osteogenic effect of residual growth factors in DBM) were implanted in surgical sites as follows: (1) DBM only (control) (n=8); (2) DBM+0.3 mg/mL Nell-1 (n=8); (3) DBM+0.6 mg/mL Nell-1 (n=8); (4) inDBM only (control) (n=4); (5) inDBM+0.3 mg/mL Nell-1 (n=4); (6) inDBM+0.6 mg/mL Nell-1 (n=4). Fusion was assessed by computed tomography, microcomputed tomography, and histology. One hundred percent fusion was achieved by 3 months in the DBM+0.6 mg/mL Nell-1 group and by 4 months in the inDBM+0.6 mg/mL Nell-1 group; bone volume and mineral density were increased by 58% and 47%, respectively. These fusion rates are comparable to published reports on BMP-2 or autograft bone efficacy in sheep. Nell-1 is an independently potent osteogenic molecule that is efficacious and easily applied when combined with DBM.

High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.

The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 µg/mL, total dose 0.375 and 0.75 µg in 75 µg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 µg/mL, total dose 2.25 µg in 75 µg total volume), and a high BMP2 concentration range (150, 300, and 600 µg/mL, total dose 11.25, 22.5, and 45 µg in 75 µg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 µg/mL.

The Nell-1 growth factor stimulates bone formation by purified human perivascular cells.

The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation.

Effect of Nell-1 delivery on chondrocyte proliferation and cartilaginous extracellular matrix deposition.

Cartilage tissue engineering using chondrogenic growth factors is an attractive strategy to promote cartilage repair. Bone morphogenetic proteins have been widely studied for their application in cartilage repair. However, functional heterogeneity of bone morphogenetic proteins and unpredictable effects such as cyst formation may limit their therapeutic use. Thus, the use of alternative growth factors with greater osteochondral specificity may be advantageous for cartilage regeneration. Nel-like molecule-1 (Nell-1; Nel is a protein strongly expressed in neural tissue encoding epidermal growth factor-like domain) is a novel growth factor believed to specifically target cells committed to the osteochondral lineage. Mutation of the Nell-1 gene has been shown to disrupt normal cartilage growth and development in rodents. This study investigates the chondrogenic potential of recombinant human Nell-1 protein in a three-dimensional alginate hydrogel microenvironment containing rabbit chondrocytes. To provide controlled delivery and maximize biological efficiency, Nell-1 was incorporated in chitosan microparticles. Over 42 days of culture, chondrocyte proliferation and cluster formation was significantly enhanced by Nell-1 in a dose-dependent manner. Further, the clusters formed in the presence of Nell-1 contained more type II collagen and glycosaminoglycans than clusters formed within Nell-free control gels. These findings demonstrate the ability of Nell-1 to promote chondrocyte proliferation and deposition of cartilage-specific extracellular matrix materials.