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In vivo biofunctional evaluation of hydrogels for disc regeneration

In vivo biofunctional evaluation of hydrogels for disc regeneration Eur Spine J (2014) 23:19–26 DOI 10.1007/s00586-013-2998-8 EUROSPI N E 2013 FULL PAPE R A WARD • • • • Sandra Reitmaier Ludwika Kreja Katharina Gruchenberg Britta Kanter • • • • Joana Silva-Correia Joaquim Miguel Oliveira Rui Luı ´s Reis Valeria Perugini • • Matteo Santin Anita Ignatius Hans-Joachim Wilke Received: 23 August 2013 / Revised: 31 August 2013 / Accepted: 31 August 2013 / Published online: 12 October 2013 The Author(s) 2013. This article is published with open access at Springerlink.com Abstract served as nucleotomy controls. 24 adult merino sheep were Purpose Regenerative strategies aim to restore the ori- used. After 6 weeks histological, after 12 weeks histolog- ginal biofunctionality of the intervertebral disc. Different ical and biomechanical analyses were conducted. biomaterials are available, which might support disc Results Biomechanical tests revealed no differences regeneration. In the present study, the prospects of success between any of the implanted and nucleotomized discs. All of two hydrogels functionalized with anti-angiogenic pep- implanted discs significantly degenerated compared to tides and seeded with bone marrow derived mononuclear intact discs. In contrast, there was no marked difference cells (BMC), respectively, were investigated in an ovine between implanted and nucleotomized discs. In tendency, nucleotomy model. albeit not significant, degeneration score and disc height Methods In a one-step procedure iliac crest aspirates index deteriorated for all but not for the cell-seeded were harvested and, subsequently, separated BMC were hydrogels from 6 to 12 weeks. Cell-seeded hydrogels seeded on hydrogels and implanted into the ovine disc. For slightly decelerated degeneration. the cell-seeded approach a hyaluronic acid-based hydrogel Conclusions None of the hydrogel configurations was was used. The anti-angiogenic potential of newly devel- able to regenerate biofunctionality of the intervertebral oped VEGF-blockers was investigated on ionically cross- disc. This might presumably be caused by hydrogel linked metacrylated gellan gum hydrogels. Untreated discs extrusion. Great importance should be given to the devel- opment of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc S. Reitmaier (&)  L. Kreja  K. Gruchenberg  B. Kanter  regeneration and restoration of intervertebral disc A. Ignatius  H.-J. Wilke functioning. Center of Musculoskeletal Research, Institute of Orthopaedic Research and Biomechanics, University of Ulm, Keywords Intervertebral disc  Degeneration Helmholtzstrasse 14, 89081 Ulm, Germany e-mail: [email protected] Regeneration  One-step  Sheep  Large animal model  In vivo J. Silva-Correia  J. M. Oliveira  R. L. Reis 3B’s Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Introduction Regenerative Medicine, AvePark, S. Cla ´udio de Barco, Taipas, Guimara ˜es, Portugal Intervertebral disc degeneration (IDD) is a highly relevant individual and socioeconomic burden, which is associated J. Silva-Correia  J. M. Oliveira  R. L. Reis ICVS/3B’s-PT Government Associate Laboratory, to various morphological and functional disturbances [1]. Braga/Guimara ˜es, Portugal The gradual progression of the disease and the structural features, e.g., the degradation of proteoglycans with sub- V. Perugini  M. Santin sequent desiccation of the disc extracellular matrix, the School of Pharmacy and Biomolecular Sciences, ingrowth of blood vessels and the loss of intervertebral disc University of Brighton, Brighton, UK 123 20 Eur Spine J (2014) 23:19–26 (IVD) height, can be addressed in close detail and predis- Configuration 2 To investigate whether anti-angiogenic pose IDD for regenerative strategies [2]. peptides reveal a positive effect in the prevention of IDD, Currently, different approaches are being pursued to iGG-MA was functionalized with non-cytotoxic polyly- regenerate the IVD. Direct injection of growth factors, viral sine-based VEGF-blockers (iGG-MA?PEP) that were vectors and cells, each alone or in combination, seek to shown to effectively inhibit endothelial cell proliferation stimulate proliferation and production of extracellular [20] (School of Pharmacy and Biomolecular Sciences, matrix [3]. Reasonable chances of success of these meth- University of Brighton, United Kingdom). ods, however, are questionable [4, 5]. Low oxygen and pH Configuration 3 Hydrogels made of dodecyl-amide of within the avascular disc represent a hostile environment hyaluronic acid (DDAHA; Anika Therapeutics, Abano for cell metabolism [2, 6, 7]. Conversely, vascularization Therme, Italy) were tested without additives. must not be promoted as it accelerates degeneration [8, 9]. Configuration 4 For the cell-based approach, DDAHA Furthermore, aberrant mechanical stimuli may activate was seeded with autologous bone marrow derived mono- catabolic remodelling, cell death and tissue breakdown nuclear cells (DDAHA?BMC). A one-step surgical pro- [10–13]. cedure was performed combining the harvesting of bone To reestablish a loading regime that enhances the ana- marrow (BM) from the iliac crest, the isolation of BMC bolic response of resident and implanted cells and to trigger and the orthotopic implantation of DDAHA?BMC. biological mechanisms of self-healing, biodegradable Configuration 5 Nucleotomy control with no treatment. substitutes were designed according to the natural ideal of The detailed composition of the hydrogels was previ- the IVD. As the restoration of disc height is assumed to be ously described [21]. essential for nucleus replacements, sufficient quantities of hydrogels are intended to be injected into the IVD for an Preparation of cell-seeded scaffolds immediate restoration of disc mechanics. Biomaterial extrusion, however, is of major biomechanical concern In 18 animals one of four operated IVD received a cell- [14–16]. Hyaluronic acid or polysaccharide-based hydro- seeded hydrogel. BM was harvested from the iliac crest gels, such as gellan gum-based hydrogels, were proven to under sterile conditions at the beginning of surgery. adequately support the growth and extracellular matrix Coagulation was prevented by 5,000 IU heparin/10 ml BM deposition of cells and might therefore be suited as nucleus (Heparin-Natrium-5,000-ratiopharm, ratiopharm GmbH, replacements [17–19]. To retain avascularity of cartilagi- Ulm, Germany). nous tissues, functionalization of scaffolds with anti- BMC (‘‘buffy coat’’) were isolated by buoyant density angiogenic peptides was suggested [20]. separation. The cell-seeded hydrogels were prepared by To gain deeper knowledge on future research directions, adding 0.25 ml of the cell suspension containing 4 9 10 the purpose of this study was to investigate newly devel- BMC/ml PBS to 0.5 ml of DDAHA, resulting in 1 9 10 oped hydrogels as nucleus replacements for the biome- BMC/DDAHA. Control samples (DDAHA) were mixed chanical restoration and biological regeneration of the disc. with 0.25 ml PBS without cells. Both hydrogels were Different modifications of hydrogels were examined in an transferred in sterile 2 ml syringes for intraoperative ovine nucleotomy model. The effect of functionalization application. For the estimation of the number of mesen- on the efficiency of hydrogels was evaluated using anti- chymal stem cells (MSC) in BMC a colony forming units- angiogenic peptides and bone marrow derived mononu- fibroblast (CFU-F) assay was performed as previously clear cells. described [22]. Orthotopic implantation of hydrogels Materials and methods All animals were operated in four levels from L1–L2 to 24 adult Merino sheep (2–4.5 years; 76–108 kg) were L4–L5 or in case of 7 lumbar vertebrae (n = 8) from L2– operated to compare four different hydrogel configurations L3 to L5–L6. General anesthesia and the retroperitoneal with nucleotomy controls. Permission for the animal multisegmental approach to the spine were performed as experiment was received from the regional commission of recently described in detail [23]. Each disc was stabbed Tu ¨ bingen (Reg. Nr. 1032). with sterile microsurgery blades and a lateral nucleotomy Configuration 1 Hydrogels made of ionic crosslinked was performed using 1.0 and 1.5 mm rongeurs with methacrylated Gellan Gum (iGG-MA; 3B’s Research Group, straight and flexed jaws, respectively. Approximately University of Minho, Portugal) were used as nucleus 0.20 g (0.17–0.23 g) nucleus tissue was removed from replacement. iGG-MA is a microbial polysaccharide, which each disc and treated in an alternating sequence with one of forms a colloidal gel in the presence of metallic ions. the five configurations (Fig. 1). The annulus defects were 123 Eur Spine J (2014) 23:19–26 21 Fig. 1 Alternating implantation scheme for the two hydrogels (blue, green) without (light) and with peptides or cells (dark) and the nucleotomy controls (red) closed with suture and 2-octyl-cyanoacrylate glue Table 1 Modified degeneration score according to Boos et al. [27] (DERMABOND , Ethicon Products, Norderstedt, Ger- Degeneration Characteristic feature Score many) and covered with a collagen sponge (Lyostypt , parameter (total 36) Aesculap AG, Tuttlingen, Germany). Intervertebral disc Eight animals were sacrificed after 6 weeks for only Cells No proliferation/ 0 histological and 16 sheep after 12 weeks for histological Increased cell density/ 1 and biomechanical analyses. The 6-week group for bio- Connection of two 2 mechanics was omitted because after this time period chondrocytes/ annulus healing is not to be expected [24]. Small size clones/ 3 For biomechanical and histological comparison with Moderate size clones/ 4 intact discs, respectively, six native lumbar spines and six Huge size clones 5 lumbar segments from independent comparable sheep were Granular changes None/rare/intermediate/ 0/1/2/3 used. abundantly Neovascularization Absent/present 0/1 Biomechanics Rim lesions None/rare/intermediate/ 0/1/2/3 abundantly For the biomechanical analyses, the polysegmental speci- Concentric tears None/rare/intermediate/ 0/1/2/3 mens including all treated discs were embedded in poly- abundantly methylmethacrylate (PMMA, Technovit 3040, Heraeus Radial tears None/rare/intermediate/ 0/1/2/3 Kulzer, Werheim, Germany) and tested in a custom-made abundantly spine loading simulator [25, 26]. The range of motion Scar formation Absent/present 0/1 (RoM) and the neutral zone (NZ) were investigated for Tissue defects Absent/present 0/1 each segment separately at ±7.5 Nm pure moments in the Endplate three principal motion planes flexion ? extension, lateral Cells None/rare/intermediate/ 0/1/2/3 abundantly bending right ? left, and axial rotation left ? right using a Structural None/rare/intermediate/ 0/1/2/3 motion tracking system (Vicon MX13, Vicon, Oxford, disorganization abundantly United Kingdom). Clefts None/rare/intermediate/ 0/1/2/3 abundantly Histology Microfracture None/rare/intermediate/ 0/1/2/3 abundantly For qualitative and semi-quantitative histological evalua- Neovascularization Absent/present 0/1 tion each segment was embedded in PMMA for undecal- New bone formation Absent/present 0/1 cified histology after formalin fixation. Giemsa Scar formation Absent/present 0/1 staining was performed using standard protocols. An Tissue defects Absent/present 0/1 established semi-quantitative degeneration score according 123 22 Eur Spine J (2014) 23:19–26 Fig. 2 Total ranges of motion (RoM) and neutral zones (NZ) in flexion ? extension (flex/ex), lateral bending right ? left (lat bend) and axial rotation left ? right (ax rot) for intact discs of separate sheep (green background) and for the five test configurations investigated in the current study. *p \ 0.05 to Boos et al. [27] was used to determine the degree of varied between 8 and 390 MSC/hydrogel. In average 0.20 ml degeneration (Table 1). The disc height index (DHI) was (0.19–0.21 ml) of the cell-seeded hydrogels could be injected. evaluated by dividing the mean value of the anterior, The estimated number of BMC injected by DDAHA ? BMC middle and posterior disc height from the histologic sam- treatment therefore was about 270,000. ples by the anterio-posterior diameter of the disc using ImageJ software [28]. Inflammation parameters, such as the Biomechanics presence of immune cells, regeneration of the tissue, blood vessel formation and remaining hydrogels were qualita- After 12 weeks in vivo, nucleotomy significantly decreased tively evaluated under light microscopy (DMI6000B, Le- RoM compared to intact discs in flexion ? extension ica, Heerbrugg, Switzerland). (p = 0.02) and lateral bending (p = 0.04; Fig. 2), but not in axial rotation. Significant decreased RoM was also found Statistics for iGG-MA ? PEP in flexion ? extension (p = 0.03) and lateral bending (p = 0.02). RoM for iGG-MA and For statistical analysis the unpaired, two-sample Wilcoxon DDAHA was significantly lower only in flexion ? exten- signed rank test to a significance level of p B 0.05 was sion (p = 0.04 and 0.01, respectively). DDAHA ? BMC used. Statistics were performed using GnuR [29]. did not show significant differences at all. Compared to nucleotomy controls none of the hydrogel implanted discs revealed significant differences. Results Histology Surgical interventions were well tolerated by the sheep. Intra- or post-operative complications did not occur. Histological sections showed marked differences between the surgically affected and intact discs 12 weeks after Cell isolation surgery. The structural integrity was obviously impaired in operated discs (Fig. 3). Tissue defects within the nucleus The yields of BMC/ml BM and the frequency of MSC were similar in size in nucleotomy controls and hydrogel evaluatedby CFU-F assay variedconsiderablybetween the implanted discs. Hydrogels could not unambiguously be sheep. The mean ± SD of BMC/ml BM was 1.11 9 10 ± identified within the histological sections. Huge size cell 0.95 9 10 . The cloning efficiency of BMC population var- clones were located in immediate vicinity of tissue defects ied between 0.0008–0.039 % CFU-F (mean 0.01 ± 0.01 %). inside the remaining nucleus pulposus tissue. Inflammation This resulted in the mean of 101 ± 108 MSC/hydrogel and could not be found in any of the investigated samples. 123 Eur Spine J (2014) 23:19–26 23 Fig. 3 Representative mid-sagittal histological sections of operated discs 12 weeks after surgery in comparison to an intact disc Fig. 5 Disc height indices of intact discs (dashed line) significantly differed from nucleotomy controls and hydrogel implanted discs Fig. 4 Degeneration scores of intact discs (dashed line) were (p = 0.003 – 0.013). No difference between nucleotomy controls and significantly less than of nucleotomy controls and hydrogel implanted the different material configurations was found both 6 (white) and discs (p = 0.003 – 0.005). There was no difference between nucle- 12 weeks (grey) post-operatively otomy controls and the four different material configurations both after 6 (white) and 12 weeks (grey) (although not significant) increase in the degeneration scores of nucleotomy controls (17 %) and all acellular test Marked neovascularisation did not occur in any of the configurations (13–35 %). In DDAHA ? BMC degenera- tion scores were kept almost constant. operated discs and, therefore, differences between the hydrogel implanted discs and the nucleotomy controls were DHI of intact discs were with 0.19 (0.16–0.25) signifi- cantly higher than all operated discs at both time intervals not found. No differences in cellularity were seen. There was no visible formation of spondylophytes or bony end- (Fig. 5). After 6 weeks DHI of nucleotomy controls fell by 28 % compared to intact discs. DHI of hydrogel implanted plate changes up to 12 weeks. discs decreased in a similar range (iGG-MA: -33 %, iGG- Intact discs revealed with a median degeneration score of 3.5 only negligible degenerative changes within the disc MApep: -30 %, DDAHA: -27 %; DDAHA ? BMC -35 %). From 6 to 12 weeks post-operatively there was a (Fig. 4). The degeneration score of all implanted and nu- cleotomized discs significantly differed from intact discs progressive decrease in DHI for acellular hydrogels (iGG- MA: -9 %, iGG-MApep: -8 %, DDAHA: -9 %). DHI in (p = 0.003–0.005). In contrast, there was no marked dif- ference between the implanted discs and nucleotomy con- DDAHA ? BMC, however, seemed to be preserved (?2 %) even if this effect was not significant. Nucleotomy trols, neither after 6 nor 12 weeks. Comparing the results after 6 to results after 12 weeks, there was an obvious controls lost 11 % of DHI between 6 and 12 weeks. 123 24 Eur Spine J (2014) 23:19–26 Discussion closed defect was additionally covered with a collagen sponge. Artificial closure devices, proven to be effective to In this study using an ovine nucleotomy model, different restore disc mechanics in vitro, unfortunately contradict to configurations of newly developed hydrogels for nucleus the main objective of disc regeneration and therefore were replacements were investigated for their ability to restore not used in the current study [33, 34]. RoM and disc height and to slow down IDD. Disc regeneration using hydrogels could not be achieved Results consistently showed that none of the tested with this ovine nucleotomy model. This is in contrast to hydrogel configurations proved to be superior to nucleot- other animal experiments with rodents or pigs [35, 36]. omy controls. The treatment with hydrogels was not able to These different findings may be explained by the persis- restore disc height. However, against expectation, the tence of notochordal progenitor cells within the discs of operated discs showed a smaller ROM than intact discs. these animals. Because in humans and likewise in sheep Histology of hydrogel implanted discs (acellular and cel- this cell type disappears, conclusions from the above lular) showed more signs of degeneration as intact discs. mentioned species should be transferred to humans with There was a slight tendency that using BMC may eventu- caution [37, 38]. Similar biology as well as similar anat- ally slow down IDD between 6 and 12 weeks of omy and biomechanics suggest the sheep to be a more implantation. relevant model for humans [39–42]. The higher RoM of intact compared to nucleotomized The potential positive effect of BMC as additive in discs found in this in vivo study is in contrast to in vitro hydrogels for disc regeneration should be interpreted data in literature, where RoM significantly increased after carefully. Only about 25 MSC/disc (range 2–70) provided nucleotomy both for human [30] and for calf specimen [14, in approximately 270,000 BMC have been implanted. The 31]. The same tendency was also found for ovine specimen optimal number of bone marrow derived MSC to be in preliminary in vitro investigations for the current study implanted into the degenerated disc to achieve a stimulat- (unpublished data). The discrepancy may be explained by ing effect was found to be 10 in dogs [43]. However, MSC general differences between in vitro conditions without yields in this order of magnitude are impossible to achieve healing, and the in vivo situation where collagenous without in vitro expansion. Low cell density applied is bridges at the outer annulus were described 12 weeks after assumed to be the reason for limited observed effects. injury [32]. This initial bridging and the formation of scar In vitro expansion of BMC prior to surgery might have tissue as well as the sealant method might be the reasons reinforced the trends. The nevertheless promising per- for the increased stiffness observed in this in vivo study. spectives of cell-based approaches for disc regeneration are The evaluation of the disc height index clearly proved in accordance to literature [44, 45]. The right cell source, that up to 12 weeks restoration of disc height could not be however, is still not finally clarified [46]. The BMC pop- achieved using different hydrogel configurations as nucleus ulation used in the current study is a broad mixture of cell replacements after nucleotomy. This finding strongly lineages in different differentiation stages containing indicates that relevant amounts of hydrogels were probably multipotent mesenchymal and hemopoietic stem cells. pressed out of the disc. This challenging problem of Beneficial effects might be mediated by differentiation of implant extrusion may also explain the similar disap- MSC into IVD cells. Furthermore, BMC may act as trophic pointing degeneration scores both for the nucleotomy factor pools. Paracrine effects may cause BMC to secrete controls as well as for the hydrogel implanted discs. growth factors, cytokines and chemokines capable of Partial removal of nucleus was required for implantation stimulating the regeneration of injured tissue [47–49]. of hydrogels as pure injection of a relevant amount of In case that the slight trend found in this study can be substitute materials into the healthy ovine disc is impos- supported with future research, the perspective of applying sible because no cavity is available and intradiscal pressure non-cultivated BMC for disc regeneration in humans might in vivo is high [23]. Opening the annulus, however, inev- be promising. Using the patients’ own and unmodified cells itably causes the problem of extrusion. Sealants that reli- might be an interesting solution which would probably lead ably keep nucleus replacements inside the disc are urgently to higher compliance of this new potential therapeutic needed, but still not available. In previous in vitro studies, option [50–52]. we have shown that the best of tested sealant options was Injected cells were not labelled in the current study. As cyanoacrylate glue combined with surgical suture [15]. with the hydrogels, the fate of BMC after injection is, Under axial compression, however, this sealant still therefore, not known. This limitation is owed to the one- allowed for gaping of the inner annulus defect with sub- step surgical procedure using BMC without in vitro sequent dislocation of implanted hydrogels and loss of cultivation. intradiscal pressure in ovine motion segments [21]. To The durations of the animal study with 6 and 12 weeks enhance the sealant efficiency in the current study, the may eventually have been too short to represent the 123 Eur Spine J (2014) 23:19–26 25 10. Ishihara H, McNally DS, Urban JP, Hall AC (1996) Effects of regenerative capacity of the different hydrogel configura- hydrostatic pressure on matrix synthesis in different regions of tions. Longer test durations are therefore recommended the intervertebral disk. J Appl Physiol 80(3):839–846 when investigating biological strategies. 11. Neidlinger-Wilke C, Wurtz K, Urban JP, Borm W, Arand M, Ignatius A, Wilke HJ, Claes LE (2006) Regulation of gene expression in intervertebral disc cells by low and high hydrostatic Conclusion pressure. Euro Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc 15(Suppl 3):S372–S378 The results of present study indicate that because of pre- 12. Fernando HN, Czamanski J, Yuan TY, Gu W, Salahadin A, sumed hydrogel extrusion, none of the investigated Huang CY (2011) Mechanical loading affects the energy metabolism of intervertebral disc cells. J Orthop Res Off Publ hydrogels was able to slow down IDD compared to nu- Orthop Res Soc 29(11):1634–1641 cleotomized discs. Therefore, the development of effective 13. Chan SC, Ferguson SJ, Gantenbein-Ritter B (2011) The effects of annulus sealants is crucial for successful nucleus replace- dynamic loading on the intervertebral disc. Eur Spine J ment using hydrogels. Additional studies are recommended 20(11):1796–1812 14. Wilke HJ, Heuer F, Neidlinger-Wilke C, Claes L (2006) Is a to substantiate the effects of non-cultivated cells for disc collagen scaffold for a tissue engineered nucleus replacement regeneration. capable of restoring disc height and stability in an animal model? Eur Spine J 15(Suppl 3):S433–S438 Acknowledgments This work was supported by the EU-project 15. Heuer F, Ulrich S, Claes L, Wilke HJ (2008) Biomechanical Disc Regeneration (NMP3-LA-2008-213904). Technical assistance of evaluation of conventional anulus fibrosus closure methods Iris Baum and the whole animal surgery team of the Institute of required for nucleus replacement. Laboratory investigation. Orthopaedic Research and Biomechanics, Ulm, are gratefully J Neurosurg Spine 9(3):307–313 acknowledged. DDAHA hydrogels were kindly provided by Cristina 16. Vadala G, Sowa G, Hubert M, Gilbertson LG, Denaro V, Kang Longinotti (DDAHA, Anika Therapeutics, Abano Therme, Italy). JD (2012) Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte forma- Conflict of interest None. tion. J Tissue Eng Regen Med 6(5):348–355 17. Revell PA, Damien E, Di Silvio L, Gurav N, Longinotti C, Open Access This article is distributed under the terms of the Ambrosio L (2007) Tissue engineered intervertebral disc repair in Creative Commons Attribution License which permits any use, dis- the pig using injectable polymers. J Mater Sci Mater Med tribution, and reproduction in any medium, provided the original 18(2):303–308 author(s) and the source are credited. 18. Silva-Correia J, Miranda-Goncalves V, Salgado A, Sousa N, Oliveira JM, Reis RM, Reis RL (2012) Angiogenic potential of gellan gum-based hydrogels for application in nucleus pulposus regeneration: In vivo study. Tissue Eng. Part A References 19. Silva-Correia J, Oliveira JM, Caridade SG, Oliveira JT, Sousa RA, Mano JF, Reis RL (2011) Gellan gum-based hydrogels for 1. Adams MA, Roughley PJ (2006) What is intervertebral disc intervertebral disc tissue-engineering applications. J Tissue Eng degeneration, and what causes it? Spine 31(18):2151–2161 Regen Med 5(6):e97–e107 2. Roberts S, Evans H, Trivedi J, Menage J (2006) Histology and 20. Meikle ST, Perugini V, Guildford AL, Santin M (2011) Synthe- pathology of the human intervertebral disc. J Bone Jt Surg Am sis, characterisation and in vitro anti-angiogenic potential of 88(Suppl 2):10–14 dendron VEGF blockers. Macromol Biosci 11(12):1761–1765 3. Paesold G, Nerlich AG, Boos N (2007) Biological treatment 21. Reitmaier S, Wolfram U, Ignatius A, Wilke HJ, Gloria A, Mar- strategies for disc degeneration: potentials and shortcomings. tinez JMM, Silva-Correia J, Oliveira JM, Reis RL,Schmidt H Euro Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur (2012) Hydrogels for nucleus replacement-facing the biome- Sect Cerv Spine Res Soc 16(4):447–468 chanical challenge. J Mech Behav Biomed Mater 4. Kandel R, Roberts S, Urban JP (2008) Tissue engineering and the 22. McCarty RC, Gronthos S, Zannettino AC, Foster BK, Xian CJ intervertebral disc: the challenges. Euro Spine J Off Publ Eur (2009) Characterisation and developmental potential of ovine Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc bone marrow derived mesenchymal stem cells. J Cell Physiol 17(Suppl 4):480–491 219:324–333 5. Chan SC, Gantenbein-Ritter B (2012) Intervertebral disc regen- 23. Reitmaier S, Schmidt H, Ihler R, Kocak T, Graf N, Ignatius eration or repair with biomaterials and stem cell therapy—fea- A,Wilke H-J (in press) Preliminary Investigations on Intradiscal sible or fiction? Swiss medical weekly 142: w13598 Pressures During Daily Activities: An In Vivo Study Using The 6. Urban JP, Smith S, Fairbank JC (2004) Nutrition of the inter- Merino Sheep. PloS one vertebral disc. Spine 29(23):2700–2709 24. Ahlgren BD, Lui W, Herkowitz HN, Panjabi MM,Guiboux JP 7. Raj PP (2008) Intervertebral disc: anatomy-physiology-patho- (2000) Effect of anular repair on the healing strength of the physiology-treatment. Pain Pract Off J World Inst Pain intervertebral disc: a sheep model. Spine (Phila Pa 1976) 25(17): 8(1):18–44 2165–2170 25. Wilke HJ, Claes L, Schmitt H, Wolf S (1994) A universal spine 8. Johnson WE, Evans H, Menage J, Eisenstein SM, El Haj A, tester for in vitro experiments with muscle force simulation. Euro Roberts S (2001) Immunohistochemical detection of Schwann cells in innervated and vascularized human intervertebral discs. Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Spine 26(23):2550–2557 Cerv Spine Res Soc 3(2):91–97 9. Kauppila LI (1995) Ingrowth of blood vessels in disc degenera- 26. Wilke HJ, Wenger K, Claes L (1998) Testing criteria for spinal tion. Angiographic and histological studies of cadaveric spines. implants: recommendations for the standardization of in vitro J Bone Jt Surg Am 77(1):26–31 stability testing of spinal implants. Eur Spine J 7(2):148–154 123 26 Eur Spine J (2014) 23:19–26 27. Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich 39. Reid JE, Meakin JR, Robins SP, Skakle JM,Hukins DW (2002) AG (2002) Classification of age-related changes in lumbar Sheep lumbar intervertebral discs as models for human discs. intervertebral discs: 2002 Volvo Award in basic science. Spine Clin Biomech (Bristol, Avon) 17(4): 312–314 27(23):2631–2644 40. Beckstein JC, Sen S, Schaer TP, Vresilovic EJ, Elliott DM (2008) 28. 1.45s I (Wayne Rasband, National Institute of Health). Available Comparison of animal discs used in disc research to human at http://imagej.nih.gov/ij lumbar disc: axial compression mechanics and glycosaminogly- 29. Team RDC (2011) R: A language and environment for statistical can content. Spine 33(6):E166–E173 computing. R Foundation for Statistical Computing, Vienna, 41. Wilke HJ, Kettler A, Claes LE (1997) Are sheep spines a valid Austria. ISBN 3-900051-07-0. Available at http://www.R-project. biomechanical model for human spines? Spine 22(20):2365–2374 org/ 42. Wilke HJ, Kettler A, Wenger KH, Claes LE (1997) Anatomy of 30. Wilke HJ, Kavanagh S, Neller S, Haid C, Claes LE (2001) Effect the sheep spine and its comparison to the human spine. Anat Rec of a prosthetic disc nucleus on the mobility and disc height of the 247(4):542–555 L4-5 intervertebral disc postnucleotomy. J Neurosurg 95(2 43. Serigano K, Sakai D, Hiyama A, Tamura F, Tanaka M, Mochida Suppl):208–214 J (2010) Effect of cell number on mesenchymal stem cell trans- 31. Hegewald AA, Knecht S, Baumgartner D, Gerber H, Endres M, plantation in a canine disc degeneration model. J Orthop Res Off Kaps C, Stussi E,Thome C (2009) Biomechanical testing of a Publ Orthop Res Soc 28(10):1267–1275 polymer-based biomaterial for the restoration of spinal stability 44. Leung VY, Chan D, Cheung KM (2006) Regeneration of inter- after nucleotomy. J Orthop Surg Res 4: 25 vertebral disc by mesenchymal stem cells: potentials, limitations, 32. Melrose J, Roberts S, Smith S, Menage J, Ghosh P (2002) and future direction. Euro Spine J Off Publ Eur Spine Soc Eur Increased nerve and blood vessel ingrowth associated with pro- Spinal Deform Soc Eur Sect Cerv Spine Res Soc 15(Suppl teoglycan depletion in an ovine anular lesion model of experi- 3):S406–S413 mental disc degeneration. Spine 27(12):1278–1285 45. Sheikh H, Zakharian K, De La Torre RP, Facek C, Vasquez A, 33. Arthur A, Cannella M, Keane M, Singhatat W, Vresilovic E, Chaudhry GR, Svinarich D, Perez-Cruet MJ (2009) In vivo Marcolongo M (2010) Fill of the nucleus cavity affects intervertebral disc regeneration using stem cell-derived chon- mechanical stability in compression, bending, and torsion of a droprogenitors. J Neurosurg Spine 10(3):265–272 spine segment, which has undergone nucleus replacement. Spine 46. Drazin D, Rosner J, Avalos P, Acosta F (2012) Stem cell therapy 35(11):1128–1135 for degenerative disc disease. Adv Orthop 2012: 961052 34. Wilke HJ, Ressel L, Heuer F, Graf N, Rath S (2013) Can pre- 47. Ren G, Chen X, Dong F, Li W, Ren X, Zhang Y, Shi Y (2012) vention of a reherniation be investigated? Establishment of a Concise review: mesenchymal stem cells and translational med- herniation model and experiments with an anular closure device. icine: emerging issues. Stem Cell Transl Med 1:51–58 Spine 38(10):E587–E593 48. Wang S, Qu X,Zhao RC (2012) Clinical applications of mesen- 35. Buser Z, Kuelling F, Liu J, Liebenberg E, Thorne KJ, Coughlin chymal stem cells. J Hematol Oncol 5: 19. Available at http:// D, Lotz JC (2011) Biological and biomechanical effects of fibrin www.jhoonline.org/content/15/11/19 injection into porcine intervertebral discs. Spine 36(18):E1201– 49. Wen Z, Zheng S, Zhou C, Wang J, Wang T (2011) Repair E1209 mechanisms of bone marrow mesenchymal cells in myocardial 36. Huang B, Zhuang Y, Li CQ, Liu LT, Zhou Y (2011) Regenera- infraction. J Cell Mol Med 15:1032–1043 tion of the intervertebral disc with nucleus pulposus cell-seeded 50. Bernardo ME, Fibbe WE (2012) Safety and efficacy of mesen- collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer con- chymal stromal cell therapy in autoimmune disorders. Ann NY structs in a rabbit disc degeneration model. Spine 36(26): Acad Sci 1266:107–117 2252–2259 51. Lepperdinger G, Brunauer R, Jamnig A, Laschober G, Kassem M 37. Hunter CJ, Matyas JR, Duncan NA (2004) Cytomorphology of (2008) Controversial issue: is it safe to employ mesenchymal notochordal and chondrocytic cells from the nucleus pulposus: a stem cells in cell-based therapies? Exp Gerontol 43(11): species comparison. J Anat 205(5):357–362 1018–1023 38. Alini M, Eisenstein SM, Ito K, Little C, Kettler AA, Masuda K, 52. So K, Takemoto M, Fujibayashi S, Neo M, Kyomoto M, Hayami Melrose J, Ralphs J, Stokes I, Wilke HJ (2008) Are animal T, Hyon SH, Nakamura T (2007) Antidegenerative effects of models useful for studying human disc disorders/degeneration? partial disc replacement in an animal surgery model. Spine Eur Spine J 17(1):2–19 32(15):1586–1591 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Spine Journal Unpaywall

In vivo biofunctional evaluation of hydrogels for disc regeneration

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Eur Spine J (2014) 23:19–26 DOI 10.1007/s00586-013-2998-8 EUROSPI N E 2013 FULL PAPE R A WARD • • • • Sandra Reitmaier Ludwika Kreja Katharina Gruchenberg Britta Kanter • • • • Joana Silva-Correia Joaquim Miguel Oliveira Rui Luı ´s Reis Valeria Perugini • • Matteo Santin Anita Ignatius Hans-Joachim Wilke Received: 23 August 2013 / Revised: 31 August 2013 / Accepted: 31 August 2013 / Published online: 12 October 2013 The Author(s) 2013. This article is published with open access at Springerlink.com Abstract served as nucleotomy controls. 24 adult merino sheep were Purpose Regenerative strategies aim to restore the ori- used. After 6 weeks histological, after 12 weeks histolog- ginal biofunctionality of the intervertebral disc. Different ical and biomechanical analyses were conducted. biomaterials are available, which might support disc Results Biomechanical tests revealed no differences regeneration. In the present study, the prospects of success between any of the implanted and nucleotomized discs. All of two hydrogels functionalized with anti-angiogenic pep- implanted discs significantly degenerated compared to tides and seeded with bone marrow derived mononuclear intact discs. In contrast, there was no marked difference cells (BMC), respectively, were investigated in an ovine between implanted and nucleotomized discs. In tendency, nucleotomy model. albeit not significant, degeneration score and disc height Methods In a one-step procedure iliac crest aspirates index deteriorated for all but not for the cell-seeded were harvested and, subsequently, separated BMC were hydrogels from 6 to 12 weeks. Cell-seeded hydrogels seeded on hydrogels and implanted into the ovine disc. For slightly decelerated degeneration. the cell-seeded approach a hyaluronic acid-based hydrogel Conclusions None of the hydrogel configurations was was used. The anti-angiogenic potential of newly devel- able to regenerate biofunctionality of the intervertebral oped VEGF-blockers was investigated on ionically cross- disc. This might presumably be caused by hydrogel linked metacrylated gellan gum hydrogels. Untreated discs extrusion. Great importance should be given to the devel- opment of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc S. Reitmaier (&)  L. Kreja  K. Gruchenberg  B. Kanter  regeneration and restoration of intervertebral disc A. Ignatius  H.-J. Wilke functioning. Center of Musculoskeletal Research, Institute of Orthopaedic Research and Biomechanics, University of Ulm, Keywords Intervertebral disc  Degeneration Helmholtzstrasse 14, 89081 Ulm, Germany e-mail: [email protected] Regeneration  One-step  Sheep  Large animal model  In vivo J. Silva-Correia  J. M. Oliveira  R. L. Reis 3B’s Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Introduction Regenerative Medicine, AvePark, S. Cla ´udio de Barco, Taipas, Guimara ˜es, Portugal Intervertebral disc degeneration (IDD) is a highly relevant individual and socioeconomic burden, which is associated J. Silva-Correia  J. M. Oliveira  R. L. Reis ICVS/3B’s-PT Government Associate Laboratory, to various morphological and functional disturbances [1]. Braga/Guimara ˜es, Portugal The gradual progression of the disease and the structural features, e.g., the degradation of proteoglycans with sub- V. Perugini  M. Santin sequent desiccation of the disc extracellular matrix, the School of Pharmacy and Biomolecular Sciences, ingrowth of blood vessels and the loss of intervertebral disc University of Brighton, Brighton, UK 123 20 Eur Spine J (2014) 23:19–26 (IVD) height, can be addressed in close detail and predis- Configuration 2 To investigate whether anti-angiogenic pose IDD for regenerative strategies [2]. peptides reveal a positive effect in the prevention of IDD, Currently, different approaches are being pursued to iGG-MA was functionalized with non-cytotoxic polyly- regenerate the IVD. Direct injection of growth factors, viral sine-based VEGF-blockers (iGG-MA?PEP) that were vectors and cells, each alone or in combination, seek to shown to effectively inhibit endothelial cell proliferation stimulate proliferation and production of extracellular [20] (School of Pharmacy and Biomolecular Sciences, matrix [3]. Reasonable chances of success of these meth- University of Brighton, United Kingdom). ods, however, are questionable [4, 5]. Low oxygen and pH Configuration 3 Hydrogels made of dodecyl-amide of within the avascular disc represent a hostile environment hyaluronic acid (DDAHA; Anika Therapeutics, Abano for cell metabolism [2, 6, 7]. Conversely, vascularization Therme, Italy) were tested without additives. must not be promoted as it accelerates degeneration [8, 9]. Configuration 4 For the cell-based approach, DDAHA Furthermore, aberrant mechanical stimuli may activate was seeded with autologous bone marrow derived mono- catabolic remodelling, cell death and tissue breakdown nuclear cells (DDAHA?BMC). A one-step surgical pro- [10–13]. cedure was performed combining the harvesting of bone To reestablish a loading regime that enhances the ana- marrow (BM) from the iliac crest, the isolation of BMC bolic response of resident and implanted cells and to trigger and the orthotopic implantation of DDAHA?BMC. biological mechanisms of self-healing, biodegradable Configuration 5 Nucleotomy control with no treatment. substitutes were designed according to the natural ideal of The detailed composition of the hydrogels was previ- the IVD. As the restoration of disc height is assumed to be ously described [21]. essential for nucleus replacements, sufficient quantities of hydrogels are intended to be injected into the IVD for an Preparation of cell-seeded scaffolds immediate restoration of disc mechanics. Biomaterial extrusion, however, is of major biomechanical concern In 18 animals one of four operated IVD received a cell- [14–16]. Hyaluronic acid or polysaccharide-based hydro- seeded hydrogel. BM was harvested from the iliac crest gels, such as gellan gum-based hydrogels, were proven to under sterile conditions at the beginning of surgery. adequately support the growth and extracellular matrix Coagulation was prevented by 5,000 IU heparin/10 ml BM deposition of cells and might therefore be suited as nucleus (Heparin-Natrium-5,000-ratiopharm, ratiopharm GmbH, replacements [17–19]. To retain avascularity of cartilagi- Ulm, Germany). nous tissues, functionalization of scaffolds with anti- BMC (‘‘buffy coat’’) were isolated by buoyant density angiogenic peptides was suggested [20]. separation. The cell-seeded hydrogels were prepared by To gain deeper knowledge on future research directions, adding 0.25 ml of the cell suspension containing 4 9 10 the purpose of this study was to investigate newly devel- BMC/ml PBS to 0.5 ml of DDAHA, resulting in 1 9 10 oped hydrogels as nucleus replacements for the biome- BMC/DDAHA. Control samples (DDAHA) were mixed chanical restoration and biological regeneration of the disc. with 0.25 ml PBS without cells. Both hydrogels were Different modifications of hydrogels were examined in an transferred in sterile 2 ml syringes for intraoperative ovine nucleotomy model. The effect of functionalization application. For the estimation of the number of mesen- on the efficiency of hydrogels was evaluated using anti- chymal stem cells (MSC) in BMC a colony forming units- angiogenic peptides and bone marrow derived mononu- fibroblast (CFU-F) assay was performed as previously clear cells. described [22]. Orthotopic implantation of hydrogels Materials and methods All animals were operated in four levels from L1–L2 to 24 adult Merino sheep (2–4.5 years; 76–108 kg) were L4–L5 or in case of 7 lumbar vertebrae (n = 8) from L2– operated to compare four different hydrogel configurations L3 to L5–L6. General anesthesia and the retroperitoneal with nucleotomy controls. Permission for the animal multisegmental approach to the spine were performed as experiment was received from the regional commission of recently described in detail [23]. Each disc was stabbed Tu ¨ bingen (Reg. Nr. 1032). with sterile microsurgery blades and a lateral nucleotomy Configuration 1 Hydrogels made of ionic crosslinked was performed using 1.0 and 1.5 mm rongeurs with methacrylated Gellan Gum (iGG-MA; 3B’s Research Group, straight and flexed jaws, respectively. Approximately University of Minho, Portugal) were used as nucleus 0.20 g (0.17–0.23 g) nucleus tissue was removed from replacement. iGG-MA is a microbial polysaccharide, which each disc and treated in an alternating sequence with one of forms a colloidal gel in the presence of metallic ions. the five configurations (Fig. 1). The annulus defects were 123 Eur Spine J (2014) 23:19–26 21 Fig. 1 Alternating implantation scheme for the two hydrogels (blue, green) without (light) and with peptides or cells (dark) and the nucleotomy controls (red) closed with suture and 2-octyl-cyanoacrylate glue Table 1 Modified degeneration score according to Boos et al. [27] (DERMABOND , Ethicon Products, Norderstedt, Ger- Degeneration Characteristic feature Score many) and covered with a collagen sponge (Lyostypt , parameter (total 36) Aesculap AG, Tuttlingen, Germany). Intervertebral disc Eight animals were sacrificed after 6 weeks for only Cells No proliferation/ 0 histological and 16 sheep after 12 weeks for histological Increased cell density/ 1 and biomechanical analyses. The 6-week group for bio- Connection of two 2 mechanics was omitted because after this time period chondrocytes/ annulus healing is not to be expected [24]. Small size clones/ 3 For biomechanical and histological comparison with Moderate size clones/ 4 intact discs, respectively, six native lumbar spines and six Huge size clones 5 lumbar segments from independent comparable sheep were Granular changes None/rare/intermediate/ 0/1/2/3 used. abundantly Neovascularization Absent/present 0/1 Biomechanics Rim lesions None/rare/intermediate/ 0/1/2/3 abundantly For the biomechanical analyses, the polysegmental speci- Concentric tears None/rare/intermediate/ 0/1/2/3 mens including all treated discs were embedded in poly- abundantly methylmethacrylate (PMMA, Technovit 3040, Heraeus Radial tears None/rare/intermediate/ 0/1/2/3 Kulzer, Werheim, Germany) and tested in a custom-made abundantly spine loading simulator [25, 26]. The range of motion Scar formation Absent/present 0/1 (RoM) and the neutral zone (NZ) were investigated for Tissue defects Absent/present 0/1 each segment separately at ±7.5 Nm pure moments in the Endplate three principal motion planes flexion ? extension, lateral Cells None/rare/intermediate/ 0/1/2/3 abundantly bending right ? left, and axial rotation left ? right using a Structural None/rare/intermediate/ 0/1/2/3 motion tracking system (Vicon MX13, Vicon, Oxford, disorganization abundantly United Kingdom). Clefts None/rare/intermediate/ 0/1/2/3 abundantly Histology Microfracture None/rare/intermediate/ 0/1/2/3 abundantly For qualitative and semi-quantitative histological evalua- Neovascularization Absent/present 0/1 tion each segment was embedded in PMMA for undecal- New bone formation Absent/present 0/1 cified histology after formalin fixation. Giemsa Scar formation Absent/present 0/1 staining was performed using standard protocols. An Tissue defects Absent/present 0/1 established semi-quantitative degeneration score according 123 22 Eur Spine J (2014) 23:19–26 Fig. 2 Total ranges of motion (RoM) and neutral zones (NZ) in flexion ? extension (flex/ex), lateral bending right ? left (lat bend) and axial rotation left ? right (ax rot) for intact discs of separate sheep (green background) and for the five test configurations investigated in the current study. *p \ 0.05 to Boos et al. [27] was used to determine the degree of varied between 8 and 390 MSC/hydrogel. In average 0.20 ml degeneration (Table 1). The disc height index (DHI) was (0.19–0.21 ml) of the cell-seeded hydrogels could be injected. evaluated by dividing the mean value of the anterior, The estimated number of BMC injected by DDAHA ? BMC middle and posterior disc height from the histologic sam- treatment therefore was about 270,000. ples by the anterio-posterior diameter of the disc using ImageJ software [28]. Inflammation parameters, such as the Biomechanics presence of immune cells, regeneration of the tissue, blood vessel formation and remaining hydrogels were qualita- After 12 weeks in vivo, nucleotomy significantly decreased tively evaluated under light microscopy (DMI6000B, Le- RoM compared to intact discs in flexion ? extension ica, Heerbrugg, Switzerland). (p = 0.02) and lateral bending (p = 0.04; Fig. 2), but not in axial rotation. Significant decreased RoM was also found Statistics for iGG-MA ? PEP in flexion ? extension (p = 0.03) and lateral bending (p = 0.02). RoM for iGG-MA and For statistical analysis the unpaired, two-sample Wilcoxon DDAHA was significantly lower only in flexion ? exten- signed rank test to a significance level of p B 0.05 was sion (p = 0.04 and 0.01, respectively). DDAHA ? BMC used. Statistics were performed using GnuR [29]. did not show significant differences at all. Compared to nucleotomy controls none of the hydrogel implanted discs revealed significant differences. Results Histology Surgical interventions were well tolerated by the sheep. Intra- or post-operative complications did not occur. Histological sections showed marked differences between the surgically affected and intact discs 12 weeks after Cell isolation surgery. The structural integrity was obviously impaired in operated discs (Fig. 3). Tissue defects within the nucleus The yields of BMC/ml BM and the frequency of MSC were similar in size in nucleotomy controls and hydrogel evaluatedby CFU-F assay variedconsiderablybetween the implanted discs. Hydrogels could not unambiguously be sheep. The mean ± SD of BMC/ml BM was 1.11 9 10 ± identified within the histological sections. Huge size cell 0.95 9 10 . The cloning efficiency of BMC population var- clones were located in immediate vicinity of tissue defects ied between 0.0008–0.039 % CFU-F (mean 0.01 ± 0.01 %). inside the remaining nucleus pulposus tissue. Inflammation This resulted in the mean of 101 ± 108 MSC/hydrogel and could not be found in any of the investigated samples. 123 Eur Spine J (2014) 23:19–26 23 Fig. 3 Representative mid-sagittal histological sections of operated discs 12 weeks after surgery in comparison to an intact disc Fig. 5 Disc height indices of intact discs (dashed line) significantly differed from nucleotomy controls and hydrogel implanted discs Fig. 4 Degeneration scores of intact discs (dashed line) were (p = 0.003 – 0.013). No difference between nucleotomy controls and significantly less than of nucleotomy controls and hydrogel implanted the different material configurations was found both 6 (white) and discs (p = 0.003 – 0.005). There was no difference between nucle- 12 weeks (grey) post-operatively otomy controls and the four different material configurations both after 6 (white) and 12 weeks (grey) (although not significant) increase in the degeneration scores of nucleotomy controls (17 %) and all acellular test Marked neovascularisation did not occur in any of the configurations (13–35 %). In DDAHA ? BMC degenera- tion scores were kept almost constant. operated discs and, therefore, differences between the hydrogel implanted discs and the nucleotomy controls were DHI of intact discs were with 0.19 (0.16–0.25) signifi- cantly higher than all operated discs at both time intervals not found. No differences in cellularity were seen. There was no visible formation of spondylophytes or bony end- (Fig. 5). After 6 weeks DHI of nucleotomy controls fell by 28 % compared to intact discs. DHI of hydrogel implanted plate changes up to 12 weeks. discs decreased in a similar range (iGG-MA: -33 %, iGG- Intact discs revealed with a median degeneration score of 3.5 only negligible degenerative changes within the disc MApep: -30 %, DDAHA: -27 %; DDAHA ? BMC -35 %). From 6 to 12 weeks post-operatively there was a (Fig. 4). The degeneration score of all implanted and nu- cleotomized discs significantly differed from intact discs progressive decrease in DHI for acellular hydrogels (iGG- MA: -9 %, iGG-MApep: -8 %, DDAHA: -9 %). DHI in (p = 0.003–0.005). In contrast, there was no marked dif- ference between the implanted discs and nucleotomy con- DDAHA ? BMC, however, seemed to be preserved (?2 %) even if this effect was not significant. Nucleotomy trols, neither after 6 nor 12 weeks. Comparing the results after 6 to results after 12 weeks, there was an obvious controls lost 11 % of DHI between 6 and 12 weeks. 123 24 Eur Spine J (2014) 23:19–26 Discussion closed defect was additionally covered with a collagen sponge. Artificial closure devices, proven to be effective to In this study using an ovine nucleotomy model, different restore disc mechanics in vitro, unfortunately contradict to configurations of newly developed hydrogels for nucleus the main objective of disc regeneration and therefore were replacements were investigated for their ability to restore not used in the current study [33, 34]. RoM and disc height and to slow down IDD. Disc regeneration using hydrogels could not be achieved Results consistently showed that none of the tested with this ovine nucleotomy model. This is in contrast to hydrogel configurations proved to be superior to nucleot- other animal experiments with rodents or pigs [35, 36]. omy controls. The treatment with hydrogels was not able to These different findings may be explained by the persis- restore disc height. However, against expectation, the tence of notochordal progenitor cells within the discs of operated discs showed a smaller ROM than intact discs. these animals. Because in humans and likewise in sheep Histology of hydrogel implanted discs (acellular and cel- this cell type disappears, conclusions from the above lular) showed more signs of degeneration as intact discs. mentioned species should be transferred to humans with There was a slight tendency that using BMC may eventu- caution [37, 38]. Similar biology as well as similar anat- ally slow down IDD between 6 and 12 weeks of omy and biomechanics suggest the sheep to be a more implantation. relevant model for humans [39–42]. The higher RoM of intact compared to nucleotomized The potential positive effect of BMC as additive in discs found in this in vivo study is in contrast to in vitro hydrogels for disc regeneration should be interpreted data in literature, where RoM significantly increased after carefully. Only about 25 MSC/disc (range 2–70) provided nucleotomy both for human [30] and for calf specimen [14, in approximately 270,000 BMC have been implanted. The 31]. The same tendency was also found for ovine specimen optimal number of bone marrow derived MSC to be in preliminary in vitro investigations for the current study implanted into the degenerated disc to achieve a stimulat- (unpublished data). The discrepancy may be explained by ing effect was found to be 10 in dogs [43]. However, MSC general differences between in vitro conditions without yields in this order of magnitude are impossible to achieve healing, and the in vivo situation where collagenous without in vitro expansion. Low cell density applied is bridges at the outer annulus were described 12 weeks after assumed to be the reason for limited observed effects. injury [32]. This initial bridging and the formation of scar In vitro expansion of BMC prior to surgery might have tissue as well as the sealant method might be the reasons reinforced the trends. The nevertheless promising per- for the increased stiffness observed in this in vivo study. spectives of cell-based approaches for disc regeneration are The evaluation of the disc height index clearly proved in accordance to literature [44, 45]. The right cell source, that up to 12 weeks restoration of disc height could not be however, is still not finally clarified [46]. The BMC pop- achieved using different hydrogel configurations as nucleus ulation used in the current study is a broad mixture of cell replacements after nucleotomy. This finding strongly lineages in different differentiation stages containing indicates that relevant amounts of hydrogels were probably multipotent mesenchymal and hemopoietic stem cells. pressed out of the disc. This challenging problem of Beneficial effects might be mediated by differentiation of implant extrusion may also explain the similar disap- MSC into IVD cells. Furthermore, BMC may act as trophic pointing degeneration scores both for the nucleotomy factor pools. Paracrine effects may cause BMC to secrete controls as well as for the hydrogel implanted discs. growth factors, cytokines and chemokines capable of Partial removal of nucleus was required for implantation stimulating the regeneration of injured tissue [47–49]. of hydrogels as pure injection of a relevant amount of In case that the slight trend found in this study can be substitute materials into the healthy ovine disc is impos- supported with future research, the perspective of applying sible because no cavity is available and intradiscal pressure non-cultivated BMC for disc regeneration in humans might in vivo is high [23]. Opening the annulus, however, inev- be promising. Using the patients’ own and unmodified cells itably causes the problem of extrusion. Sealants that reli- might be an interesting solution which would probably lead ably keep nucleus replacements inside the disc are urgently to higher compliance of this new potential therapeutic needed, but still not available. In previous in vitro studies, option [50–52]. we have shown that the best of tested sealant options was Injected cells were not labelled in the current study. As cyanoacrylate glue combined with surgical suture [15]. with the hydrogels, the fate of BMC after injection is, Under axial compression, however, this sealant still therefore, not known. This limitation is owed to the one- allowed for gaping of the inner annulus defect with sub- step surgical procedure using BMC without in vitro sequent dislocation of implanted hydrogels and loss of cultivation. intradiscal pressure in ovine motion segments [21]. To The durations of the animal study with 6 and 12 weeks enhance the sealant efficiency in the current study, the may eventually have been too short to represent the 123 Eur Spine J (2014) 23:19–26 25 10. Ishihara H, McNally DS, Urban JP, Hall AC (1996) Effects of regenerative capacity of the different hydrogel configura- hydrostatic pressure on matrix synthesis in different regions of tions. Longer test durations are therefore recommended the intervertebral disk. J Appl Physiol 80(3):839–846 when investigating biological strategies. 11. Neidlinger-Wilke C, Wurtz K, Urban JP, Borm W, Arand M, Ignatius A, Wilke HJ, Claes LE (2006) Regulation of gene expression in intervertebral disc cells by low and high hydrostatic Conclusion pressure. Euro Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc 15(Suppl 3):S372–S378 The results of present study indicate that because of pre- 12. Fernando HN, Czamanski J, Yuan TY, Gu W, Salahadin A, sumed hydrogel extrusion, none of the investigated Huang CY (2011) Mechanical loading affects the energy metabolism of intervertebral disc cells. J Orthop Res Off Publ hydrogels was able to slow down IDD compared to nu- Orthop Res Soc 29(11):1634–1641 cleotomized discs. Therefore, the development of effective 13. Chan SC, Ferguson SJ, Gantenbein-Ritter B (2011) The effects of annulus sealants is crucial for successful nucleus replace- dynamic loading on the intervertebral disc. Eur Spine J ment using hydrogels. Additional studies are recommended 20(11):1796–1812 14. Wilke HJ, Heuer F, Neidlinger-Wilke C, Claes L (2006) Is a to substantiate the effects of non-cultivated cells for disc collagen scaffold for a tissue engineered nucleus replacement regeneration. capable of restoring disc height and stability in an animal model? Eur Spine J 15(Suppl 3):S433–S438 Acknowledgments This work was supported by the EU-project 15. Heuer F, Ulrich S, Claes L, Wilke HJ (2008) Biomechanical Disc Regeneration (NMP3-LA-2008-213904). Technical assistance of evaluation of conventional anulus fibrosus closure methods Iris Baum and the whole animal surgery team of the Institute of required for nucleus replacement. Laboratory investigation. Orthopaedic Research and Biomechanics, Ulm, are gratefully J Neurosurg Spine 9(3):307–313 acknowledged. DDAHA hydrogels were kindly provided by Cristina 16. Vadala G, Sowa G, Hubert M, Gilbertson LG, Denaro V, Kang Longinotti (DDAHA, Anika Therapeutics, Abano Therme, Italy). JD (2012) Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte forma- Conflict of interest None. tion. J Tissue Eng Regen Med 6(5):348–355 17. Revell PA, Damien E, Di Silvio L, Gurav N, Longinotti C, Open Access This article is distributed under the terms of the Ambrosio L (2007) Tissue engineered intervertebral disc repair in Creative Commons Attribution License which permits any use, dis- the pig using injectable polymers. J Mater Sci Mater Med tribution, and reproduction in any medium, provided the original 18(2):303–308 author(s) and the source are credited. 18. Silva-Correia J, Miranda-Goncalves V, Salgado A, Sousa N, Oliveira JM, Reis RM, Reis RL (2012) Angiogenic potential of gellan gum-based hydrogels for application in nucleus pulposus regeneration: In vivo study. Tissue Eng. Part A References 19. Silva-Correia J, Oliveira JM, Caridade SG, Oliveira JT, Sousa RA, Mano JF, Reis RL (2011) Gellan gum-based hydrogels for 1. Adams MA, Roughley PJ (2006) What is intervertebral disc intervertebral disc tissue-engineering applications. J Tissue Eng degeneration, and what causes it? Spine 31(18):2151–2161 Regen Med 5(6):e97–e107 2. Roberts S, Evans H, Trivedi J, Menage J (2006) Histology and 20. Meikle ST, Perugini V, Guildford AL, Santin M (2011) Synthe- pathology of the human intervertebral disc. J Bone Jt Surg Am sis, characterisation and in vitro anti-angiogenic potential of 88(Suppl 2):10–14 dendron VEGF blockers. Macromol Biosci 11(12):1761–1765 3. Paesold G, Nerlich AG, Boos N (2007) Biological treatment 21. Reitmaier S, Wolfram U, Ignatius A, Wilke HJ, Gloria A, Mar- strategies for disc degeneration: potentials and shortcomings. tinez JMM, Silva-Correia J, Oliveira JM, Reis RL,Schmidt H Euro Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur (2012) Hydrogels for nucleus replacement-facing the biome- Sect Cerv Spine Res Soc 16(4):447–468 chanical challenge. J Mech Behav Biomed Mater 4. Kandel R, Roberts S, Urban JP (2008) Tissue engineering and the 22. McCarty RC, Gronthos S, Zannettino AC, Foster BK, Xian CJ intervertebral disc: the challenges. Euro Spine J Off Publ Eur (2009) Characterisation and developmental potential of ovine Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc bone marrow derived mesenchymal stem cells. J Cell Physiol 17(Suppl 4):480–491 219:324–333 5. Chan SC, Gantenbein-Ritter B (2012) Intervertebral disc regen- 23. Reitmaier S, Schmidt H, Ihler R, Kocak T, Graf N, Ignatius eration or repair with biomaterials and stem cell therapy—fea- A,Wilke H-J (in press) Preliminary Investigations on Intradiscal sible or fiction? Swiss medical weekly 142: w13598 Pressures During Daily Activities: An In Vivo Study Using The 6. Urban JP, Smith S, Fairbank JC (2004) Nutrition of the inter- Merino Sheep. PloS one vertebral disc. Spine 29(23):2700–2709 24. Ahlgren BD, Lui W, Herkowitz HN, Panjabi MM,Guiboux JP 7. Raj PP (2008) Intervertebral disc: anatomy-physiology-patho- (2000) Effect of anular repair on the healing strength of the physiology-treatment. Pain Pract Off J World Inst Pain intervertebral disc: a sheep model. Spine (Phila Pa 1976) 25(17): 8(1):18–44 2165–2170 25. Wilke HJ, Claes L, Schmitt H, Wolf S (1994) A universal spine 8. Johnson WE, Evans H, Menage J, Eisenstein SM, El Haj A, tester for in vitro experiments with muscle force simulation. Euro Roberts S (2001) Immunohistochemical detection of Schwann cells in innervated and vascularized human intervertebral discs. Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Spine 26(23):2550–2557 Cerv Spine Res Soc 3(2):91–97 9. Kauppila LI (1995) Ingrowth of blood vessels in disc degenera- 26. Wilke HJ, Wenger K, Claes L (1998) Testing criteria for spinal tion. Angiographic and histological studies of cadaveric spines. implants: recommendations for the standardization of in vitro J Bone Jt Surg Am 77(1):26–31 stability testing of spinal implants. Eur Spine J 7(2):148–154 123 26 Eur Spine J (2014) 23:19–26 27. Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich 39. Reid JE, Meakin JR, Robins SP, Skakle JM,Hukins DW (2002) AG (2002) Classification of age-related changes in lumbar Sheep lumbar intervertebral discs as models for human discs. intervertebral discs: 2002 Volvo Award in basic science. Spine Clin Biomech (Bristol, Avon) 17(4): 312–314 27(23):2631–2644 40. Beckstein JC, Sen S, Schaer TP, Vresilovic EJ, Elliott DM (2008) 28. 1.45s I (Wayne Rasband, National Institute of Health). Available Comparison of animal discs used in disc research to human at http://imagej.nih.gov/ij lumbar disc: axial compression mechanics and glycosaminogly- 29. Team RDC (2011) R: A language and environment for statistical can content. Spine 33(6):E166–E173 computing. R Foundation for Statistical Computing, Vienna, 41. Wilke HJ, Kettler A, Claes LE (1997) Are sheep spines a valid Austria. ISBN 3-900051-07-0. Available at http://www.R-project. biomechanical model for human spines? Spine 22(20):2365–2374 org/ 42. Wilke HJ, Kettler A, Wenger KH, Claes LE (1997) Anatomy of 30. Wilke HJ, Kavanagh S, Neller S, Haid C, Claes LE (2001) Effect the sheep spine and its comparison to the human spine. Anat Rec of a prosthetic disc nucleus on the mobility and disc height of the 247(4):542–555 L4-5 intervertebral disc postnucleotomy. J Neurosurg 95(2 43. Serigano K, Sakai D, Hiyama A, Tamura F, Tanaka M, Mochida Suppl):208–214 J (2010) Effect of cell number on mesenchymal stem cell trans- 31. Hegewald AA, Knecht S, Baumgartner D, Gerber H, Endres M, plantation in a canine disc degeneration model. J Orthop Res Off Kaps C, Stussi E,Thome C (2009) Biomechanical testing of a Publ Orthop Res Soc 28(10):1267–1275 polymer-based biomaterial for the restoration of spinal stability 44. Leung VY, Chan D, Cheung KM (2006) Regeneration of inter- after nucleotomy. J Orthop Surg Res 4: 25 vertebral disc by mesenchymal stem cells: potentials, limitations, 32. Melrose J, Roberts S, Smith S, Menage J, Ghosh P (2002) and future direction. Euro Spine J Off Publ Eur Spine Soc Eur Increased nerve and blood vessel ingrowth associated with pro- Spinal Deform Soc Eur Sect Cerv Spine Res Soc 15(Suppl teoglycan depletion in an ovine anular lesion model of experi- 3):S406–S413 mental disc degeneration. Spine 27(12):1278–1285 45. Sheikh H, Zakharian K, De La Torre RP, Facek C, Vasquez A, 33. Arthur A, Cannella M, Keane M, Singhatat W, Vresilovic E, Chaudhry GR, Svinarich D, Perez-Cruet MJ (2009) In vivo Marcolongo M (2010) Fill of the nucleus cavity affects intervertebral disc regeneration using stem cell-derived chon- mechanical stability in compression, bending, and torsion of a droprogenitors. J Neurosurg Spine 10(3):265–272 spine segment, which has undergone nucleus replacement. Spine 46. Drazin D, Rosner J, Avalos P, Acosta F (2012) Stem cell therapy 35(11):1128–1135 for degenerative disc disease. Adv Orthop 2012: 961052 34. Wilke HJ, Ressel L, Heuer F, Graf N, Rath S (2013) Can pre- 47. Ren G, Chen X, Dong F, Li W, Ren X, Zhang Y, Shi Y (2012) vention of a reherniation be investigated? Establishment of a Concise review: mesenchymal stem cells and translational med- herniation model and experiments with an anular closure device. icine: emerging issues. Stem Cell Transl Med 1:51–58 Spine 38(10):E587–E593 48. Wang S, Qu X,Zhao RC (2012) Clinical applications of mesen- 35. Buser Z, Kuelling F, Liu J, Liebenberg E, Thorne KJ, Coughlin chymal stem cells. J Hematol Oncol 5: 19. Available at http:// D, Lotz JC (2011) Biological and biomechanical effects of fibrin www.jhoonline.org/content/15/11/19 injection into porcine intervertebral discs. Spine 36(18):E1201– 49. Wen Z, Zheng S, Zhou C, Wang J, Wang T (2011) Repair E1209 mechanisms of bone marrow mesenchymal cells in myocardial 36. Huang B, Zhuang Y, Li CQ, Liu LT, Zhou Y (2011) Regenera- infraction. J Cell Mol Med 15:1032–1043 tion of the intervertebral disc with nucleus pulposus cell-seeded 50. Bernardo ME, Fibbe WE (2012) Safety and efficacy of mesen- collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer con- chymal stromal cell therapy in autoimmune disorders. Ann NY structs in a rabbit disc degeneration model. Spine 36(26): Acad Sci 1266:107–117 2252–2259 51. Lepperdinger G, Brunauer R, Jamnig A, Laschober G, Kassem M 37. Hunter CJ, Matyas JR, Duncan NA (2004) Cytomorphology of (2008) Controversial issue: is it safe to employ mesenchymal notochordal and chondrocytic cells from the nucleus pulposus: a stem cells in cell-based therapies? Exp Gerontol 43(11): species comparison. J Anat 205(5):357–362 1018–1023 38. Alini M, Eisenstein SM, Ito K, Little C, Kettler AA, Masuda K, 52. So K, Takemoto M, Fujibayashi S, Neo M, Kyomoto M, Hayami Melrose J, Ralphs J, Stokes I, Wilke HJ (2008) Are animal T, Hyon SH, Nakamura T (2007) Antidegenerative effects of models useful for studying human disc disorders/degeneration? partial disc replacement in an animal surgery model. Spine Eur Spine J 17(1):2–19 32(15):1586–1591

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European Spine Journal – Unpaywall

Published: Oct 12, 2013

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