Mohawk is a transcription factor that promotes meniscus cell phenotype and tissue repair and reduces osteoarthritis severity.

Meniscus tears are common knee injuries and a major osteoarthritis (OA) risk factor. Knowledge gaps that limit the development of therapies for meniscus injury and degeneration concern transcription factors that control the meniscus cell phenotype. Analysis of RNA sequencing data from 37 human tissues in the Genotype-Tissue Expression database and RNA sequencing data from meniscus and articular cartilage showed that transcription factor Mohawk (MKX) is highly enriched in meniscus. In human meniscus cells, MKX regulates the expression of meniscus marker genes, OA-related genes, and other transcription factors, including Scleraxis (SCX), SRY Box 5 (SOX5), and Runt domain-related transcription factor 2 (RUNX2). In mesenchymal stem cells (MSCs), the combination of adenoviral MKX (Ad-MKX) and transforming growth factor-ß3 (TGF-ß3) induced a meniscus cell phenotype. When Ad-MKX-transduced MSCs were seeded on TGF-ß3-conjugated decellularized meniscus scaffold (DMS) and inserted into experimental tears in meniscus explants, they increased glycosaminoglycan content, extracellular matrix interconnectivity, cell infiltration into the DMS, and improved biomechanical properties. Ad-MKX injection into mouse knee joints with experimental OA induced by surgical destabilization of the meniscus suppressed meniscus and cartilage damage, reducing OA severity. Ad-MKX injection into human OA meniscus tissue explants corrected pathogenic gene expression. These results identify MKX as a previously unidentified key transcription factor that regulates the meniscus cell phenotype. The combination of Ad-MKX with TGF-ß3 is effective for differentiation of MSCs to a meniscus cell phenotype and useful for meniscus repair. MKX is a promising therapeutic target for meniscus tissue engineering, repair, and prevention of OA.

FOXO1 and FOXO3 transcription factors have unique functions in meniscus development and homeostasis during aging and osteoarthritis.

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.

In Vitro and In Vivo Performance of Tissue-Engineered Tendons for Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful; autografts or allografts have certain disadvantages; and synthetic grafts have had poor clinical results. PURPOSE: To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow-derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to those experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes and newly synthesized collagen and glycosaminoglycan were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The ultimate tensile load to failure and stiffness of recellularized constructs were measured after dynamic stimulation. Finally, the tissue-engineered tendons were used to reconstruct the ACL in 24 pigs, and ultimate tensile load to failure and stiffness were assessed after 3 months. RESULTS: Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and glycosaminoglycan, and the ultimate tensile load and stiffness of recellularized tendons. After in vivo reconstruction, the ultimate tensile load and stiffness of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the ultimate tensile load of the natural ACL. CONCLUSION: This translational study indicates that recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and ultimate tensile load of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts. CLINICAL RELEVANCE: Dynamic bioreactor cultivation of tissue-engineered tendons may overcome the limitations of autografts and allografts.

Herniated intervertebral disk induces hypertrophy and ossification of ligamentum flavum.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). OBJECTIVES: To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. METHODS: LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzyme-linked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. RESULTS: Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1α, interleukin-6, tumor necrosis factor-α, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. CONCLUSIONS: Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines.

Inflammatory cytokines induce fibrosis and ossification of human ligamentum flavum cells.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and various inflammatory cytokines. OBJECTIVES: To examine the effect of inflammatory cytokines on LF cells and to identify their roles in the pathogenesis of LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by the degenerative processes (ie, increased collagen synthesis and chondroid metaplasia) of ligament fibroblasts. Degenerated intervertebral disk spontaneously produces inflammatory cytokines, which might affect the adjacent LF through local milieu of the spinal canal. METHODS: The interlaminar portion of the LF was collected during surgical spinal procedures in 15 patients (age range, 49-78 y) with lumbar spinal stenosis. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with various inflammatory cytokines: interleukin (IL)-1α, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO). Cytotoxicity was analyzed by MTT assays. DNA synthesis was measured with H-thymidine incorporation, and mRNA expression of types I, III, V, and XI collagen and osteocalcin were performed by reverse transcription-polymerase chain reaction. Histochemical stains such as Von Kossa were also performed to detect bone nodule formation. RESULTS: There was no cytotoxicity in the LF cells treated with each cytokine. There were significant increases in DNA synthesis and upregulated mRNA expression of types I, V, XI collagen and osteocalcin in LF cultures treated with various cytokines. LF cultures treated with IL-6, TNF-α, PGE2, and NO showed positive Von Kossa staining, indicating bone nodule formation from LF cells. CONCLUSIONS: Inflammatory cytokines (IL-6, TNF-α, PGE2, and NO) seem to play a crucial role in hypertrophy and ossification of LF. Degenerated, herniated intervertebral disks, and facet arthrosis may influence LF through inflammatory cytokines and cause hypertrophy and ossification of LF.

Osteoinductive activity of biphasic calcium phosphate with different rhBMP-2 doses in rats.

OBJECTIVE: The aim of the current study was to determine whether a hydroxyapatite (HA)/beta-tricalcium phosphate (ß-TCP) ratio of 20/80 impregnated with recombinant human bone morphogenetic protein (rhBMP-2) enhances new bone formation and to evaluate the dose-dependent response of rhBMP-2. STUDY DESIGN: Critical-sized calvarial defects were made in rats, and biphasic calcium phosphate (BCP) with different rhBMP-2 doses was loaded into rat calvarial defects. The animals were allowed to heal for either 2 or 8 weeks. RESULTS: The percentages of new bone after 2 and 8 weeks of healing were significantly greater in the rhBMP-2-treated groups (at all doses) than in the control groups. The percentage of remaining BCP was significantly lower at 8 weeks than at 2 weeks in all groups that included BCP. CONCLUSIONS: rhBMP-2 administered using a BCP carrier significantly induces new bone formation. A dose-dependent response was not shown in the present study.

The risk assessment of a fall in patients with lumbar spinal stenosis.

STUDY DESIGN: A prospective case control study. OBJECTIVES: To investigate the risk of a fall by using functional mobility tests in patients with lumbar spinal stenosis (LSS) via a comparison with patients with knee osteoarthritis (KOA). SUMMARY OF BACKGROUND DATA: LSS is a degenerative arthritic disease in the spine that results in decreasing function, impaired balance, and gait deficit, with increased levels of leg and back pain. This physical impairment may result in an increased risk of fall later in the disease process, as shown in KOA. However, there has been no study regarding the association between the risk of a fall and LSS. METHODS: The study was an age- and weight-matched case control study consisting of two groups: one group consisting of 40 patients with LSS who were scheduled to undergo spine surgery (LSS group) and the other group consisting of 40 patients with advanced osteoarthritis in both knees, scheduled to undergo TKA on both knees (KOA group). For both groups, four functional mobility tests, such as a Six-Meter-Walk Test (SMT), Sit-to-Stand test (STS), Alternative-Step Test (AST), and Timed Up and Go Test (TUGT), were performed. RESULTS: There was no difference in demographic data between both groups except for body mass index. For the SMT and STS, the patients in the LSS group spent significantly more time performing these tests than the patients in the KOA. For the AST, however, patients in the KOA group presented a statistically worse performance in functional mobility, compared with the LSS group. The mean TUGT time was not statistically different between the two groups. CONCLUSIONS: The current study highlights that patients with symptomatic LSS have a risk of a fall comparable with the patients who had degenerative KOA based on the results of functional mobility tests (SMT, STS, AST, and TUGT).

De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer.

STUDY DESIGN: In vitro and in vivo experiment using degenerated human ligamentum flavum (LF) and Type 5 adenovirus construct with bone morphogenetic protein-2 (BMP-2) cDNA. OBJECTIVES: To demonstrate in vitro and in vivo osteogenic effect of BMP-2 gene transfer to human LF and to propose genetically modified LF as a substitute for autogenous bone graft in spinal fusion. SUMMARY OF BACKGROUND DATA: Spinal fusion is still considered to be an important option for treating various spinal disorders. To induce solid spinal fusion, osteoinductive and/or osteoconductive agents have been widely adopted. Autogenous LF, however, has never been seriously considered as a carrier for ex vivo osteoinductive gene therapy for spinal fusion. METHODS: In vitro experiment: Degenerated human LF was harvested and cultured. Type 5 adenovirus lacZ (Ad/lacZ) and BMP-2 construct (Ad/BMP-2) were produced. LF cell cultures were then exposed to Ad/BMP-2. Expressions of osteocalcin and BMP-2 mRNA were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis was performed to detect osteocalcin protein. Alkaline phosphatase and von Kossa stains were used to detect osteogenic markers and bone nodule formation, respectively. In vivo experiment: Human LF tissues treated with Ad/lacZ, Ad/BMP-2, and saline were implanted into the subcutaneous tissue of nude mice. After 4 weeks, nude mice were radiographed and killed. Implanted LF tissues were harvested and histologically stained. RESULTS: LF cell cultures with Ad/BMP-2 revealed strong expression of BMP-2 and osteocalcin mRNA in RT-PCR and osteocalcin protein in western blot analysis. LF cell culture with saline showed baseline expression of BMP-2, osteocalcin mRNA, and osteocalcin protein, respectively. Furthermore, LF cell culture with Ad/BMP-2 demonstrated the expression of alkaline phosphatase and bone nodule formation in the aforementioned histochemical stain. LF tissues with Ad/BMP-2 revealed de novo osteogenesis in nude mice, whereas LF with Ad/lacZ or saline showed only remaining LF tissue without sign of bone formation. CONCLUSION: Human LF cells transduced with Ad/BMP-2 exhibited the expression of osteogenic phenotype and bone nodule formation. Additionally, genetically modified human LF with BMP-2 cDNA clearly demonstrated de novo osteogenesis, which supports the concept that biologically modified LF can be a substitute for autogenous bone graft in spinal fusion surgery.

Radiographic and functional outcome after surgical management of severe scoliosis in skeletally immature patients with muscular dystrophy.

OBJECTIVE: Most patients with muscular dystrophy (MD) develop progressive scoliosis after losing ambulatory status, but some cases develop severe scoliosis at a skeletally immature age before losing ambulatory status. Only a few studies have been conducted in skeletally immature patients with severe scoliosis. The purpose of this study was to assess the functional and cosmetic outcome in skeletally immature patients with severe scoliosis. METHODS: Preoperative, immediate postoperative, and final follow-up radiographs were analyzed in 10 consecutive skeletally immature patients with respect to the Cobb angle degree and the pelvic obliquity angle correction, how long the correction was maintained, and the development of the crankshaft phenomenon. In the functional assessment, the ability to sit balanced, according to the Mulcahy method, and the ability to use hands, according to the Rhyu method, were evaluated. Furthermore, the degree of subjective satisfaction was evaluated in these patients. RESULTS: The average age of the patients was 10.4 years, and the average follow-up period was 33 months with minimum 2 years' follow-up. All 10 patients survived and were available at the follow-up. The mean Cobb and pelvic obliquity angles were 80 degrees and 17 degrees at the time of the surgery, 31 degrees and 3.7 degrees immediately after the surgery, and 35 degrees and 4.7 degrees at the time of the final follow-up, respectively. The initial mean Cobb angle correction averaged 61%, with 78% of pelvic obliquity corrected. These corrections were maintained over time in most cases. At the time of the surgery, the mean volume of blood loss was 1111 mL, with an average operation time of 411 minutes. There were no major complications. At the time of the last follow-up, no patient showed development of the crankshaft phenomenon. The average score for the ability to sit balanced improved from 4.4 to 6.6 according to the Mulcahy evaluation method. The scores for hand use were 2.2-2.7. However, the forced vital capacity of the lungs decreased from a preoperative 48% to 46.1%. CONCLUSIONS: These results indicate that even in very young MD patients with severe scoliosis, acceptable curve correction can be achieved and maintained with surgery. The improved pelvic obliquity and scoliosis angle stabilized the spine, freeing the upper extremities and allowing productive activities characteristic of childhood.