Allogeneic Mesenchymal Progenitor Cells for Posterolateral Lumbar Spine Fusion in Sheep

Authors

Donna L. Wheeler, PhD, Joseph M. Lane, MD, Howard B. Seim III, DVM, Dipl. ACVS, Christian Puttlitz, PhD, Silviu Itescu, MD, A. Simon Turner, B.V.Sc, M.S, Dipl. ACVS

Abstract

Background

Osteoconductive porous ceramic bone graft materials supplemented with mesenchymal precursor cells (MPC) derived from autologous bone marrow aspirates have been shown to stimulate successful interbody and posterolateral spine fusion in preclinical models. Recent advances in immunomagnetic cell sorting have enabled purification and isolation of pleuripotent stem cells from marrow aspirates and have expanded stem cell technology to allogeneic cell sources. Allogeneic MPC technology combined with appropriate synthetic biomaterial carriers could provide both the osteogenic and osteoconductive components needed for successful posterolateral spine fusion without the need for autologous bone harvest or expensive recombinant protein technology.

Purpose

To determine the safety and efficacy of a hydroxyapatite:tricalcium phosphate graft material supplemented with allogeneic mesenchymal precursor cells in posterolateral lumbar spine fusion using an ovine model.

Study Design

Skeletally mature ewes underwent single-level instrumented posterolateral lumbar spine fusion using either autograft (AG), hydroxyapatite:tricalcium phosphate carrier (CP), or CP supplemented with allogeneic mesenchymal progenitor cells (MPCs). Three doses of MPCs were evaluated: 25 X 106 cells (low dose, LD), 75 X 106 cells (mid dose, MD), and 225 X 106 cell (high dose, HD). Animals survived for either 4 or 9 months.

Methods

Plain radiographs were acquired and scored for bridging bone at regular intervals during healing to monitor fusion development. Hematology, coagulation and serum chemistry were monitored at regular interval throughout the study to monitor animal health. After necropsy, computed tomography, high resolution radiography, biomechanical testing, organ pathology, bone histopathology, and bone histomorphometry were conducted to monitor the safety and ascertain the efficacy of MPC treatment.

Results

MPC treatment in this spine fusion model resulted in no observed adverse systemic or local tissue responses. Radiographically, fusion scores for MPC treated animals were uniformly higher compared to those treated with carrier alone (CP) after 3 months and continued the same trend throughout 9 month of healing. Quantitative computed tomography (qCT) confirmed better connectivity of the fusion for MPC treatment groups compared to CP. Biomechanical analyses were not able to differentiate between treatment groups. Histomorphometry results confirmed radiographic and qCT results; cell-supplemented treatment groups and autograft had equivalent amounts of bone within the fusion mass and less bony fusion tissue was found within the fusion mass in specimens from the CP treatment group. No conclusive effects of cell dose of fusion efficacy were noted.

Conclusions

Adult allogeneic mesenchymal precursor cells delivered via a hydroxyapatite:tricalcium phosphate carrier were both safe and efficacious in this ovine spine fusion model. Results from this preclinical study support that allogeneic mesenchymal precursor cells produced fusion efficacy similar to that achieved using iliac crest autograft, thereby providing a safe and viable option to achieve successful posterolateral spine fusion.

Link to Article

http://dx.doi.org/10.1016/j.spinee.2013.09.048

Treatment with resveratrol attenuates sublesional bone loss in spinal-cord-injured rats

Authors

Hua-Dong Wang, Ya-Min Shi, Li Li, Ji-Dong Guo, Yu-Peng Zhang, Shu-Xun Hou

Abstract

Background and purpose: Sublesional osteoporosis predisposes individuals with spinal cord injury (SCI) to an increased risk of low-trauma fracture. The aim of present work was to investigate the effect of treatment with resveratrol (RES) on sublesional bone loss in spinal-cord-injured rats. Experimental approach: Complete SCI was generated by surgical transaction of the cord at the T10-12 level. Treatment with RES (400 mg/kg body mass/day, intragastrically) was initiated 12 hours after the surgery for 10 days. Then, blood was collected and femurs and tibiae were removed for evaluation of the effects of RES on bone tissue after SCI. Key results: Treatment of SCI rats with RES prevented the reduction of bone mass including bone mineral content and bone mineral density in tibiae, preserved bone structure including trabecular bone volume fraction, trabecular number, and trabecular thickness in tibiae, and preserved mechanical strength including ultimate load, stiffness, and energy in femurs. Treatment of SCI rats with RES enhanced femoral t-SH content, reduced femoral malondialdehyde and IL-6 mRNA levels. Treatment of SCI rats with RES suppressed the upregulation of mRNA levels of PPARγ, aP2, and LPL, and restored mRNA levels of Wnt1, Lrp5, Axin2, ctnnb1, IGF-1 and IGF-1R in femurs and tibiae. Conclusions and Implications: Treatment with RES attenuated sublesional bone loss in spinal-cord-injured rats, associated with abating oxidative stress, attenuating inflammation, depressing PPARγ signaling, and restoring Wnt/β-catenin and IGF-1 signaling.

Link to Article

http://dx.doi.org/10.1111/bph.12301

Bone remodelling and aposition onto calcium phosphate coated polymer surfaces in the spine: an in vivo animal model

Authors

T Steffen, BJC Freeman and M Aebi

Abstract

Long term, secondary implant fixation of Total Disc Replacements (TDR) can be enhanced by hydroxyapatite or similar osseo-conductive coatings. These coatings are routinely applied to metal substrates. The objective of this in vivo study was to investigate the early stability and subsequent bone response adjacent to an all polymer TDR implant over a period of six months in an animal model. Six skeletally mature male baboons (Papio annubis) were followed for a period of 6 months. Using a transperitoneal exposure, a custom-sized Cadisc L device was implanted into the disc space one level above the lumbo-sacral junction in all subjects. Radiographs of the lumbar spine were acquired prior to surgery, and post-operatively at intervals up to 6 months to assess implant stability. Flourochrome markers (which contain molecules that bind to mineralization fronts) were injected at specified intervals in order to investigate bone remodeling with time. Animals were humanely euthanized six months after index surgery. Test and control specimens were retrieved, fixed and subjected to histological processing to assess the bone-implant-bone interface. Fluorescence microscopy and confocal scanning laser microscopy were utilized with BioQuant image analysis to determine the bone mineral apposition rates and gross morphology. Radiographic evaluation revealed no loss of disc height at the operative level or adjacent levels. No evidence of subsidence or significant migration of the implant up to 6 months. Heterotopic ossification was observed to varying degrees at the operated level. Histology revealed the implant primary fixation features embedded within the adjacent vertebral endplates. Flourochrome distribution revealed active bone remodeling occurring adjacent to the polymeric end-plate with no evidence of adverse biological responses. Mineral apposition rates of between 0.7 and 1.7 microns / day are in keeping with literature values for hydroxyapatite coated implants in cancellous sites of various species. Radiographic assessment demonstrates that the Cadisc L implant remains stable in vivo with no evidence of subsidence or significant migration. Histological analysis suggests the primary fixation features are engaged, and in close apposition with the adjacent vertebral bone. Flourochrome markers provide evidence of a positive bone remodelling response in the presence of the implant.

Link to Article

http://www.bjjprocs.boneandjoint.org.uk/content/94-B/SUPP_XXXVI/85.short

Ex Vivo Transfer of the Hoxc-8-interacting Domain of Smad1 by a Tropism-modified Adenoviral Vector Results in Efficient Bone Formation in a Rabbit Model of Spinal Fusion

Authors

Douglas, J.T. and Rivera, A.A. and Lyons, G.R. and Lott, P.F. and Wang, D. and Zayzafoon, M. and Siegal, G.P. and Cao, X. and Theiss, S.M.

Abstract

Study Design: Ex vivo gene transfer for spinal fusion.

Objective: This study aimed to evaluate ex vivo transfer of the nuclear-localized Hoxc-8-interacting domain of Smad1 (termed Smad1C) to rabbit bone marrow stromal cells (BMSCs) by a tropism-modified human adenovirus serotype 5 (Ad5) vector as a novel therapeutic approach for spinal fusion.

Summary of Background Data: Novel approaches are needed to improve the success of bone union after spinal fusion. One such approach is the ex vivo transfer of a gene encoding an osteoinductive factor to BMSCs which are subsequently reimplanted into the host. We have previously shown that heterologous expression of the Hoxc-8-interacting domain of Smad1 in the nuclei of osteoblast precursor cells is able to stimulate the expression of genes related to osteoblast differentiation and induce osteogenesis in vivo. Gene delivery vehicles based on human Ad5 are well suited for gene transfer for spinal fusion because they can mediate high-level, short-term gene expression. However, Ad5-based vectors with native tropism poorly transduce BMSCs, necessitating the use of vectors with modified tropism to achieve efficient gene transfer.

Methods: The gene encoding Smad1C was transferred to rabbit BMSCs by an Ad5 vector with native tropism or a vector retargeted to αv integrins, which are abundantly expressed on rabbit BMSCs. Transduced BMSCs were maintained in osteoblastic differentiation medium for 30 days. Alkaline phosphatase activity was determined and cells stained for calcium deposition. As positive controls for osteogenesis, we used Ad5 vectors expressing bone morphogenetic protein 2. As negative controls, BMSCs were mock-transduced or transduced with an Ad5 vector expressing β-galactosidase. In an immunocompetent rabbit model of spinal fusion, transduced BMSCs were coated onto absorbable gelatin sponge and implanted between decorticated transverse processes L6 and L7 of 8-week-old female New Zealand white rabbits. Animals were killed 4 weeks after implantation of the sponges, the fusion masses harvested and the area of new bone quantified using image analysis software.

Results: The Smad1C-expressing tropism-modified Ad5 vector mediated a significantly higher level of alkaline phosphatase activity and calcium deposition in transduced rabbit BMSCs than all other vectors. The rabbit BMSCs transduced ex vivo with the Smad1C-expressing tropism-modified Ad5 vector mediated a greater amount of new bone formation than BMSCs transduced with any other vector.

Conclusions: Delivery of the Smad1C gene construct to BMSCs by an αv integrin-targeted Ad5 vector shows promise for spinal fusion and other applications requiring the formation of new bone in vivo.

Link to Article

http://journals.lww.com/jspinaldisorders/Abstract/2010/02000/Ex_Vivo_Transfer_of_the_Hoxc_8_interacting_Domain.13.aspx