Ashish D. Diwan

The biology of bone grafting.

Abstract Many approaches are used to repair skeletal defects in reconstructive orthopaedic surgery, and bone grafting is involved in virtually every procedure. The type of bone graft used depends on the clinical scenario and the anticipated final outcome. Autogenous cancellous bone graft, with its osteogenic, osteoinductive, and osteoconductive properties, remains the standard for grafting. However, the high incidence of morbidity during autogenous graft harvest may make the acquisition of grafts from other sources desirable. The clinical applications for each type of bone graft are dictated by the structure and biochemical properties of the graft. An elegant cellular and molecular cascade follows bone transplantation. Bone graft incorporation within the host, whether autogenous or allogeneic, depends on many factors: type of graft (autogenous versus allogeneic, vascular versus nonvascular), site of transplant, quality of transplanted bone and host bone, host bed preparation, preservation techniques, systemic and local disease, and mechanical properties of the graft.

Osteoporosis influences the early period of fracture healing in a rat osteoporotic model

Osteoporotic fractures commonly occur in the elderly. Although current therapies are aimed at the prevention and treatment of osteoporotic fractures, studies examing the fracture healing process in osteoporotic bone are limited. We produced an osteoporotic rat model by ovariectomy (ovx) and maintained a low calcium diet (LCD) in order to evaluate the influence of osteoporosis on fracture healing. Callus formation and strength was monitored over a 3 week period by histological and biomechanical assessment. Data collected simultaneously on a group of rats undergoing sham surgery (sx) were used for comparison. A 40% reduction in fracture callus cross-sectional area and a 23% reduction in bone mineral density in the healing femur of the ovx rats was observed on day 21 following fracture as compared with the sx group (p < 0.01). Biomechanical data from the healing femur of the ovx rats revealed a fivefold decrease in the energy required to break the fracture callus, a threefold decrease in peak failure load, a twofold decrease in stiffness and a threefold decrease in stress as compared with the sx group (p < 0.01, respectively). Histomorphological analysis revealed a delay in fracture callus healing with poor development of mature bone in the ovx rats. This study provides physical evidence of altered fracture healing in osteoporotic bone, which may have important implications in evaluating the effects of new treatments for osteoporosis on fracture healing.

Recurrent and Injurious Falls in the Year Following Hip Fracture: A Prospective Study of Incidence and Risk Factors From the Sarcopenia and Hip Fracture Study

BACKGROUND The incidence and etiology of falls in patients following hip fracture remains poorly understood. METHODS We prospectively investigated the incidence of, and risk factors for, recurrent and injurious falls in community-dwelling persons admitted for surgical repair of minimal-trauma hip fracture. Fall surveillance methods included phone calls, medical records, and fall calendars. Potential predictors of falls included health status, quality of life, nutritional status, body composition, muscle strength, range of motion, gait velocity, balance, walking endurance, disability, cognition, depression, fear of falling, self-efficacy, social support, physical activity level, and vision. RESULTS 193 participants enrolled in the study (81 +/- 8 years, 72% women, gait velocity 0.3 +/- 0.2 m/s). We identified 227 falls in the year after hip fracture for the 178 participants with fall surveillance data. Fifty-six percent of participants fell at least once, 28% had recurrent falls, 30% were injured, 12% sustained a new fracture, and 5% sustained a new hip fracture. Age-adjusted risk factors for recurrent and injurious falls included lower strength, balance, range of motion, physical activity level, quality of life, depth perception, vitamin D, and nutritional status, and greater polypharmacy, comorbidity, and disability. Multivariate analyses identified older age, congestive heart failure, poorer quality of life, and nutritional status as independent risk factors for recurrent and injurious falls. CONCLUSIONS Recurrent and injurious falls are common after hip fracture and are associated with multiple risk factors, many of which are treatable. Interventions should therefore be tailored to alleviating or reversing any nutritional, physiological, and psychosocial risk factors of individual patients.

Nitric oxide modulates fracture healing.

The role of the messenger molecule nitric oxide has not been evaluated in fracture healing. NO is synthesized by three kinds of nitric oxide synthase (NOS): inducible NOS (iNOS), endothelial (eNOS), and neuronal (bNOS). We evaluated the role of these enzymes in a rat femur fracture‐healing model. There was no messenger RNA (mRNA) expression, immunoreactivity, or enzymatic activity for NOS in unfractured femoral cortex. After fracture, however, mRNA, protein, and enzymatic activity for iNOS were identified in the healing rat femoral fracture callus, with maximum activity on day 15. The mRNA expression for eNOS and bNOS was induced slightly later than for iNOS, consistent with a temporal increase in calcium‐dependent NOS activity that gradually increased up to day 30. mRNA expression for the three NOS isoforms also was found in six of six human fracture callus samples. To study the effect of suppression of NO synthesis on fracture healing, an experimental group of rats was fed an NOS inhibitor, l‐nitroso‐arginine methyl ester (l‐NAME), and the control group was fed its inactive enantiomer, d‐nitroso‐arginine methyl ester (d‐NAME). An 18% (p ≤ 0.01) decrease in cross‐sectional area and a 45% (p ≤ 0.05) decrease in failure load were observed in the NOS‐inhibited group on day 24 after fracture. Furthermore, the effect of NO supplementation to fracture healing was studied by delivering NO to the fracture site using carboxybutyl chitosan NONOate locally. On day 17 after fracture, there was a 30% (p ≤ 0.05) increase in cross‐sectional area in the NO‐donor group compared with the NOS inhibition group. These results show for the first time that NO is expressed during fracture healing in rats and in humans, that suppression of NOS impairs fracture healing, and that supplementation of NO can reverse the inhibition of healing produced by NOS inhibitors. (J Bone Miner Res 2000;15:342–351)

Mutations in GDF6 are associated with vertebral segmentation defects in Klippel-Feil syndrome.

Klippel‐Feil syndrome (KFS) is a congenital disorder of spinal segmentation distinguished by the bony fusion of anterior/cervical vertebrae. Scoliosis, mirror movements, otolaryngological, kidney, ocular, cranial, limb, and/or digit anomalies are often associated. Here we report mutations at the GDF6 gene locus in familial and sporadic cases of KFS including the recurrent missense mutation of an extremely conserved residue c.866T>C (p.Leu289Pro) in association with mirror movements and an inversion breakpoint downstream of the gene in association with carpal, tarsal, and vertebral fusions. GDF6 is expressed at the boundaries of the developing carpals, tarsals, and vertebrae and within the adult vertebral disc. GDF6 knockout mice are best distinguished by fusion of carpals and tarsals and GDF6 knockdown in Xenopus results in a high incidence of anterior axial defects consistent with a role for GDF6 in the etiology, diversity, and variability of KFS. Hum Mutat 0,1–11, 2008.

Histologic evaluation of the efficacy of rhBMP-2 compared with autograft bone in sheep spinal anterior interbody fusion.

Study Design. The sheep anterior lumbar spinal fusion model was used to study the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2)–collagen composite in comparison with autograft to enhance spinal interbody fusion. Comparisons were drawn from temporal radiographic and end-point biomechanical and histologic data. Objective. To analyze histologically the ability of rhBMP-2 to achieve complete arthrodesis between vertebral bodies. Summary of Background Data. Studies using rhBMP for enhancement of anterior interbody fusion have used numerous endpoints. However, systematic histologic evaluation of the fusion has not been conducted. Methods. Twelve sheep underwent single-level anterior lumbar interbody fusion performed with a cylindrical fenestrated titanium interbody fusion device (INTER FIX, Medtronic Sofamor Danek, Inc., Memphis, TN). The device was filled either with rhBMP-2–collagen (n = 6) or autogenous iliac crest bone graft (n = 6). Radiologic evaluation was carried out at 2-month intervals, and all sheep were killed 6 months after surgery. Nondestructive biomechanical testing for stiffness to flexion, extension, and lateral bending moments, un-decalcified histology, and qualitative and quantitative histologic evaluation were performed. Results. Radiographs revealed a bony bridge anterior to the cage in five of six rhBMP-2-treated animals, whereas it was present only in one of five in the autogenous bone graft group. Segments treated with rhBMP-2 were 20% stiffer in flexion than autograft-treated segments at 6 months. Six of six in the rhBMP-2 group and two of six in the autograft group showed complete fusion. There was a significantly higher rate of bony continuity observed at the fenestrations of the rhBMP-2 group. Three times more number of cage fenestrations in the rhBMP-2 group demonstrated “all-bone” when compared with the autograft group (P < 0.001). Further, the scar tissue in and around the autograft-treated cages was 16-fold more (P < 0.01) than that seen for rhBMP-2-treated cages. Conclusions. The study demonstrates that rhBMP-2 can lead to earlier radiologic fusion and a more consistent increased stiffness of the segments when compared with autograft in sheep anterior lumbar interbody fusion. Furthermore, a three times higher histologic fusion rate is attainable with significantly reduced fibrous tissue around the implant when rhBMP-2 is used.

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010.

BMP-2 Enhances TGF-β3–Mediated Chondrogenic Differentiation of Human Bone Marrow Multipotent Mesenchymal Stromal Cells in Alginate Bead Culture

This study addresses synergistic effects of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta3 (TGF-beta3) in the induction of chondrocytic differentiation of bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro for potential use in intervertebral disc (IVD) repair. Human BM MSCs encapsulated in alginate beads were induced to differentiate in serum-free medium containing BMP-2 and TGF-beta3. The expression of chondrocytic genes and proteins was analyzed by real-time PCR, western blot, histological, and immunohistochemical assays. This differentiation system showed a potent induction of chondrocytic phenotypes. The expression of chondrocytic markers, such as aggrecan (ACAN) and type II collagen (COL2A1), was upregulated at higher levels than using TGF-beta3 alone. Blocking BMP-2 by noggin completely suppressed BMP-2-enhanced gene and protein expression, confirming a crucial input of BMP-2 signaling in this differentiation process. Inhibition of extracellular signal-regulated kinases 1 and 2 signaling resulted in an increase in ACAN and COL2A1 gene expression, suggesting a negative regulatory role of this pathway. In conclusion, BMP-2 enhances TGF-beta3-mediated chondrogenesis of MSCs. The combination of BMP-2 and TGF-beta3 in alginate culture is superior to the standard differentiation method using TGF-beta alone. This potent induction system may provide an alternative cell source for IVD and cartilage regeneration in clinical practice.

Heterogeneity in Klippel-Feil syndrome: a new classification

Background. Klippel-Feil syndrome (KFS) is characterised by congenital vertebral fusion of the cervical spine and a wide spectrum of associated anomalies. KFS has often been considered a sporadic syndrome. However, since the publication of the original KFS classification early this century, a number of KFS families have indicated heterogeneity complicated by a broad range of variable expression. Objective. The two major objectives of this study were (1) to identify differences and similarities in the postnatal appearance, morphology, position and inheritance of vertebral fusions within and between KFS families and (2) to establish a new KFS classification focussed on KFS aetiology. Materials and methods. Vertebral fusions were assessed via spinal radiography. Chromosomal karyotypes were performed using routine cytogenetics. Results. The medical histories of three KFS families are presented. The postnatal time, position and appearance of vertebral fusions, associated anomalies and mode of inheritance were different for the three KFS families. Four classes of KFS are described in a comprehensive classification table that allays much of the uncertainty arising from KFS heterogeneity and variable expression. Conclusion. We have described four different KFS classes (KF1–4) within a comprehensive classification that addresses KFS genetic heterogeneity. The position of vertebral fusions in the cervical spine and their incidence within affected families are delineating features of KFS.

Cell therapy for disc degeneration—potentials and pitfalls

Disc degeneration is considered a major source of pain in patients with chronic low back pain. Novel strategies to cure or decrease the symptoms and increase the patients quality of life and function are under development. Until recently conservative treatment and fusion surgery were the main therapeutic options. Disc prostheses are undergoing clinical evaluation. The potential for cell transplantation to the intervertebral disc with mature autologous disc cells, chondrocytes, or stem cells is in early stages of investigation. Cell transplantation potentially can increase proteoglycan production and induce disc regeneration or slow down the degeneration process. In animal models, transplantation of autologous disc cells and chondrocytes (derived from costal cartilage) has been demonstrated to be feasible and may slow disc degeneration.