Hegewald Et Al-2013-Journal of Tissue Engineering and Regenerative Medicine

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Enhancing tissue repair in annulus brosus defectsof the intervertebral disc: analysis of a bio-integrativeannulus implant in an in-vivo ovine model Aldemar Andres Hegewald 1,2 *, Fabian Medved 1 , Daxiong Feng 1,3 , Charalambos Tsagogiorgas 4 , Anja Beierfu 5 , Genevieve Ama Kyremaa Schindler 1 , Marcus Trunk 6 , Christian Kaps 7,Demissew Shenegelegn Mern 1 and Claudius Thom 21 Department of Neurosurgery, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany 2 Department of Neurosurgery, Innsbruck Medical University, Innsbruck, Austria3

Spinal Surgery Department, The Af liated Hospital of Luzhou Medical College, Luzhou, Sichuan Province, P R China4 Department of Anaesthesiology and Critical Care Medicine, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany 5 Department of Small Animal Surgery and Ophthalmology, Ludwig Maximilian s University Munich, Munich, Germany 6 Institute of Pathology, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany 7 TransTissue Technologies GmbH, Berlin, Germany

Abstract

Annulus brosus repair techniques for the intervertebral disc (IVD) address the unsolved problem of reherniation after IVD herniation and might facilitate the development of nucleus pulposus replace-ment techniques for IVD diseases. This study investigates the suitability of a bio-integrative annulus

implant.Standardized box defects were applied to the annulus L3/4 and L4/5 of 16 sheep, followedby randomized insertion of the textile polyglycolic acid/polyvinylidene uoride annulus implant inone of the defects. Explantation was conducted after 2, 6 and 12 weeks, followed by provocativepressure testing and histological analysis. At 2 weeks follow-up, all specimens of the control defectgroupdemonstrated uncontained herniated nucleuspulposustissue in the annulusdefects.For the treatedspecimens, the annulus implant consistently provided an effective barrier for herniating nucleus pulposustissue, with no implant dislocation at all time-points. After 2 weeks, a homogeneous cell in ltration of theannulus implant was observed, leading to a progressive directional matrix build-up. Repair tissue thick-ness was signi cantly stronger with the annulus implant at all follow-ups ( p < 0.01). No pronouncedforeign body reaction and no difference in the amount of supra-annular scar tissue over the defect sites were observed. The implantation procedure in icted annulus damage adjacent to the defect. At latertime-points, however, no difference in comparison with the control defect group was evident. The investi-gated biointegrative annulus implant showed promising results with regard to biointegration, enhance-ment of repair tissue and function as a mechanical barrier in an ovine model. 2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

Received 3 October 2012; Revised 5 May 2013; Accepted 2 September 2013

Keywords annulus brosus; biomaterial; disc herniation; intervertebral disc; regeneration; spine surgery

1. Introduction

Annulus brosus (AF) repair techniques for theintervertebral disc (IVD) are of interest to the spine sur-geon because (1) they address the unsolved problem of

*Correspondence to: Aldemar Andres Hegewald, Department of Neurosurgery, University Medical Centre Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1 3, 68167 Mannheim, Germany. E-mail: [email protected]

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modi cations or adaptations are made.

2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med (2013)Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1831
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reherniation through the untreated annulus defectafter IVD herniation (Barth et al ., 2008) and (2) they facilitate the development of nucleus pulposus re-placement techniques for IVD diseases by providingadequate nucleus containment (Heuer et al., 2008).Both are highly relevant health economic issues

Sherman et al ., 2010).First attempts of AF closure consisted of simple suturing

and gluing techniques (Ahlgren et al., 2000; Heuer et al.,2008) and of the insertion of solid mechanical barriers(Bron et al., 2010; Anulex Technologies, 2012; IntrinsicTherapeutics, 2012). These techniques were only partly successful and were often complicated by implant disloca-tion. Moreover, there is the concern of long-term effects of solid implants on adjacent tissue structures. New genera-tions of these implants are now in the process of con-trolled clinical trials.

Advances in material sciences and biotechnologicaltechniques are now encouraging innovative biologicalrepair strategies. Biological scaffold technology playsa major role in providing primary stability and three-dimensional spaces for tissue formation by introduc-ing a whole array of absorbable and non-absorbablebiomaterials (Ratner and Bryant, 2004; Chan andLeong, 2008). In addition, bioactive factors, enforcingcell proliferation and matrix production (Gruber et al .,1997, Imai et al., 2007, Gilbertson et al., 2008, Kim et al.,2009, Vadal et al., 2012) as well as cell chemotaxis(Hegewald et al., 2011), might enhance the AF repairprocess. Cell-based approaches with multipotent progenitorcells or chondrogenic cell lines are discussed as possible

therapeutic options (Kuh et al., 2009; Koepsell et al., 2011)and might open the door for gene therapy approaches(Zhang et al., 2007).

This pilot study reports on the suitability of a cell-free, biointegrative annulus implant to enhance tissuerepair in a large box-shaped AF defect by means of an in-vivo ovine model. The potential advantages of this implant are its high porosity and its highbioabsorbability.

The natural repair process of AF defects advancesfrom the outside to the inside, but rarely exceeds theouter third of the defect, leaving the inner regions

of the AF unrepaired (Key and Ford, 1948; Smithand Walmsley, 1951; Hampton e t al., 1989; Ethieret al ., 1994; Melrose et al., 2008). This nding andthe authors experience in biomechanical studies that AF implants demonstrate a certain amount of bulgingunder load simulation (Hegewald et al ., 2009) lead tothe hypothesis of this study that an optimal implanta-tion site for an AF implant is at the inner wall of the AF, allowing some bulging into the AF defect withoutcompromising the spinal canal. At the same time, the AF implant can exert its function as a biological scaf-fold by enabling repair processes in the inner regionsof the AF, otherwise left unrepaired. In this way, theestablishment of a more reliable AF repair process with enhanced primary stability and repair tissuestrength is expected.

2. Methods

2.1. Annulus implant

A customized triphase AF implant 10 mm wide, 5 mmhigh and 2 mm thick, consisting of two outer phases of absorbable polyglycolic acid (PGA) nonwoven and acentric phase of a non-absorbable polyvinylidene uoride(PVDF) mesh was fabricated and assembled. The PGA (Purac Biomaterials, Gorinchem, the Netherlands) wasmelt spun to multi lament bres and processed to anon-woven, characterized by a porosity of 80% and aninterconnecting pore network, feasible for cell in-growthand matrix build-up. The microstructure of the nonwoven consists of short laments arranged in all direc-tions and bonded together mechanically using needlepunching. The stabilizing textile mesh is made of non-absorbable PVDF bers with a diameter of 112 m

(G. Krahmer GmbH, Buchholz, Germany). This textilemesh enforces primary stability, especially at the xationpoints of the implant. The mesh was produced using adouble raschel warp knitting machine (DDR 16 EAC/EEC;Karl Mayer Textilmaschinenfabrik GmbH, Obertshausen,Germany). In a nal step, the warp knitted PVDF mesh(centric phase) and the doubled PGA non-woven (two outerphases) were assembled by customized needle punching.

2.2. Surgical procedure

Twenty healthy adult merino sheep, aged 4 6 years,

mean body weight 82 kg, were operated on within thisproject with the approval of the local competentauthority (Regierungsprsidium Karlsruhe, le number35-9185.81/G-15/09).

The rst two sheep were operated via a dorsal inter-laminar approach. In contrast to the human situs, it wasfound that the outgoing spinal nerve roots are not protectedby a dural sleeve and are therefore very sensitive to mobiliza-tion. Both sheep were killed post-operatively because of high-grade hind-limb weakness, and a left lateral retro-peritoneal approach was then adopted for the study.Two sheep developed a severe wound infection and had

to be killed prematurely. Consequently, 16 sheep wereincluded in the study. After suitable intramuscular preliminary medication

with xylazine (0.1 0.15 mg/kg body weight) and keta-mine (10 mg/kg body weight), the sheep was intubatedand a large stomach tube applied. The maintenance of narcosis was provided by inhalation anaesthesia withiso urane (1.5 3 vol %) and supported by intravenousfentanyl (1 3 g/kg body weight) if required. Periopera-tive antibiotic prophylaxis was provided with intravenouscefazolin (2 g). Intraoperative monitoring was established with arterial blood pressure measurement and measure-ment of oxygen saturation.

The sheep was carefully bedded on the right side. Usingstandard sterile techniques, the lumbar segments L3/4and L4/5 were accessed via a left retroperitoneal, transpsoas

A. A. Hegewald et al.

2013 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013)DOI: 10.1002/term


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(between psoas major and minor) approach. After uoro-scopic examination for level localization, two box-shapedannulus defects of 3.5 3.5 mm were applied with a scalpelto the two intervertebral discs. Protruding nucleus pulposustissue in thedefect wascarefully coagulated andtheadjacent AF was undermined with a hook instrument. The nucleus

pulposus was otherwise left intact and no nucleotomy was performed. A blocked randomization for the annu-lus implant insertion was then conducted.

Directly before application, the annulus implant wasimmersed in autologous blood serum for potentialbioactivation (Hegewald et al., 2011). The implantation was achieved with an inside-out xation technique, usingnon-absorbable sutures (4-0 Prolene , ETHICON GmbH,Norderstedt, Germany) that were pre xed at the four cor-ners of the annulus implant (Figure1). With this technique,the annulus implant could be pulled inside the disc andtightly xed to the inner AF.

2.3. Explantation procedure and provocativepressure testing

Explantation was performed 2, 6 and 12 weeks aftersurgery with ve, ve and six sheep, respectively, in eachexplantation group. For each explanted lumbar spine, block randomization between the IVDs L2/3 and L5/6 wasconducted to allocate for: (a) fresh annulus defect, (b) fresh

annulus defect with implant and (c) intact control. In theanalysis, (a) and (b) are referred to as time-point 0 weeks.

Following this procedure, provocative pressure testing was performed. Under uoroscopic observation, a cus-tomized high-pressure needle was placed centrally intothe nucleus pulposus. With an angiography pump, contin-

uous injection (0.5ml/min) of contrast medium wasapplied to the nucleus pulposus compartment until con-trast media leakage was observed uoroscopically. A pressure transducer was used to continuously registerthe pressure values.

2.4. Histology

Tissue processing and staining protocols were mostly basedon procedures established by Melrose et al. (2004, 2007).

Directly after provocative pressure testing, individualIVDs were excised with a band saw and xed in 10%neutral buffered formalin (1 week). Decalci cation wasperformed in several changes (three times a week) of 10% formic acid in 5% neutral buffered formalin withconstant agitation (4 weeks). After decalci cation, thespecimens were rinsed in running tap water (30 min)and equilibrated in several changes of 70% ethanol.To prepare tissue sections, specimens were double-embedded in paraf n celloidin. Specimens were rstdehydrated in graded alcohols and afterwards

Figure 1. (a) Anterolateral view of an annulus implant inserted directly after explantation of the spine in an annulus box defect. (b,c)Top and axial view, respectively, of the inside-out xation technique of the annulus implant. After treatment with methyl-benzoate,

the pellucid appearance of this 12-week specimen reveals the four xation points of the sutures in the annulus implant, the suturing technique from the inside to the outside of the disc and the position of the implant within the annulus brosus. (d f) Demonstrationof the intraoperative implantation procedure via a retroperitoneal approach: (d) application of an annulus box defect; (e) pulling theannulus implant inside the disc with preset sutures; (f) completed implantation and adjacent control defect

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equilibrated in methyl-benzoate for 24 h. To in ltratethe tissue with celloidin, specimens were incubated ina mixture of celloidin and methylbenzoate. First, they were equilibrated in methylbenzoate containing 1%celloidin, then subsequently in methylbenzoate containing5% celloidin. After puri cation with chloroform for 1 hour,

the specimens were vacuum-in ltrated and embedded inParaplast (Paraplast Plus , Sigma-Aldrich , Germany). Be-fore cutting, thesurface was softened by using softening so-lution containing ammonia, glycerine, 100% ethanol anddistilled water. Afterwards, 10 m sections were cut witha microtome and xed on star frost plus (SuperFrost PlusObjekttrger, R. Langenbrinck Labor- und Medizintechnik,Emmendingen, Germany) glass slides. Sections were driedat 75C on a heating plate (10 min) and then overnight inan oven at 55C. Deparaf nization was performed in xylene.Thereafter, specimens were rehydrated with graded ethanol washes (100 70% v/v).

The following stains were performed: (1) modi edMasson s trichrome, (2) toluidine blue fast green, bothaccording to modi ed protocols established by Melroseet al . (2007), (3) FAST (Fast Green, Alcian Blue,Safranin-O, tartrazine) according to modi ed protocolsof Leung et al. (2009). For the Masson s trichromestaining, the sections were rst incubated in Bouin smordant xative at 60C (1 h) and afterwards puri edin running tap water (10 min). Sections were stained with Weigert-s iron-haematoxylin reagent (30 min);adjacent colour differentiation was performed in 1%HCl in 70% ethanol. Afterwards, sections were puri edin running tap water and Scott s tap water substitute

(3 min) and stained in Ponceau de Xylidine (8 min). After being washed with distilled water, sections weredifferentiated in 5% phosphotungstic acid solution(10 min) and counterstained with 1% Fast GreenFCF (2 min). Finally, sections were dehydrated in severalchanges of absolute ethanol and cleared in xylene. WithMasson s trichrome, cytoplasm and muscle bres stainedred and collagen bres stained blue. Somewhat unexpected was the consistent reddish staining of the AF; this wasalso found in the repair tissue of all 12-weeks specimens.For Toluidine Blue Fast Green staining, sections werestained in 0.04% Toluidine Blue O (10 min) and thencounterstained with 0,1% Fast Green FCF (2 min),

puri ed with several changes of isopropanol and clearedin xylene. With Toluidine Blue Fast Green staining,proteoglycans are stained blue. For multichromaticFAST (Fast Green, Alcian Blue, Safranin-O, tartrazine)staining, initial staining was performed with AlcianBlue 1% (2.5 min) and Safranin-O 0.1% (3 min). Afterdifferentiation with ethanol 50% (1 min) counterstaining was performed with tartrazine 0.25% (10 s) and Fast Green0.001% (5 min). Finally, sections were dehydrated inabsolute ethanol and cleared in xylene. With FASTstaining, acidic glycoproteins stained blue, neutralglycoproteins appeared orange and brous tissueappeared yellowish. After each staining and differenti-ation step, sections were brie y rinsed in distil led water and mounted in eukitt (EUKITT , O. KindlerGmbH, Freiburg, Germany) after the staining proce-dure was completed.

2.5. Histological analysis

Histological analysis was performed with assistance of thedigital image processing software for microscopesAxiovision Version 4.8.1 (Zeiss; AxioVision, Carl Zeiss Microscopy GmbH, Gttingen, Germany). Two investigators analysed a

total of 57 intervertebral disc specimens. A mean of 17 (SD3.7) axial sections from the centre of the intervertebral disc were analysed according to the criteria outlined in (Table 1).

2.6. Statistical analysis

Data analysis was performed with GRAPHPAD PRISM version5.0d for Mac OS X (GraphPad Software, San Diego, CA,USA). Quantitative data from both groups at single time-points were compared using the nonparametric Mann

Whitney test (two-tailed). For data comparison at differ-

ent time-points, the non-parametric Kruskal

Wallis test with Dunn s post test was applied. Categorical variables were analysed with Fisher s exact test (two-sided). Thesigni cance level for all the statistical tests was p = 0.05.

3. Results

Sixteen sheep were included in the study. They did not showany anaesthesia or surgically related complications. There were no device-relatedor other health-related complications.

Contrast media leakage with provocative pressure

testing directly after explantation was observed at a meanpressure of 0.53 MPa, with high variance within thegroups. There were no statistical signi cant differencesbetween groups after 2, 6 and 12 weeks. Similarly, there were no differences within groups at different time-points. The intact control did not show any contrast medialeakage up to the pressure limit of 4.8 MPa of the system.

At the time-points 0 weeks and 2 weeks,all 10 specimensof the control defect group demonstrated uncontainedherniated nucleus pulposus tissue in the annulus defects(Figure 2c,d). For the treated specimens, the annulusimplant consistently provided an effective barrier for herni-

ating nucleus pulposus tissue (Figure2d,e) with no implantdislocation at all time-points. After 6 weeks and 12 weeks,both groups showed comparable results with regard toclosing the annulus defect.

The implantation procedure in icts annulus damageadjacent to the defect in comparison to sole injury of theannulus (Figure 2f). At later time-points, however, nodifferences were evident.

After 2 weeks, a homogeneous cell in ltration, includingthe central parts of the annulus implant, is observed(Figure 3a,b). After 6 weeks, the absorbable component(PGA) of the annulus implant completely degradedand degradation products could only sporadically befound in some specimens (Figure 3c,d). The remainingnon-absorbable component (PVDF) was found to besteeped by repair tissue that impressed with directional




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bre bundles. After 12 weeks, the density of the extra-cellular matrix considerably increased and a progressive

attening of the cells could be observed (Figure 3e,f).Repair tissue thickness was signi cantly greater in the

annulus implant group at all follow-ups (Figure 4a,b,d,e,g). After 2 weeks, the annulus implant group demonstrateda moderate biological integration with the host tissue

(Figure 4i). With increasing follow-up, the stability andamount of repair tissue integration improved in bothgroups (Figure 4c,f,i). No pronounced foreign body reac-tion or granuloma formation was observed. Repair tissuein both groups showed similar appearances, althoughthe repair tissue in the defect group appeared to bemore highly vascularized in most specimens (Figure 4f).There was no difference in the amount of supra-annularscar tissue over the defect site between the two groups(Figure 4h).

At 6 weeks follow-up, the control defect groupdisplayed higher proportions of Toluidine Blue stainingthat were site-concordant with bluish staining in FAST,indicating high proteoglycan proportions in the repairtissue (Figure 5c). In contrast, at later follow-up, FastGreen staining dominated the repair tissue in both groups

(Figure 5a c), which was site-concordant with yellow-orange staining in FAST, indicating a more broustissue with lower proteoglycan concentrations.

The 6-week follow-up was accompanied by the pro-nounced appearance of disc cell proliferation clusters inthe nucleus pulposus of both groups (Figure 5d f), indi-cating a degenerative process. These cell clusters were

predominately located at the base of the annulus defect.

4. Discussion

Early reports on repair mechanisms of AF defects havealready described the formation of a brous cap in theouter regions of the defects, leaving the inner regionsunrepaired (Key and Ford, 1948; Smith and Walmsley,1951). Further studies reported on the speci c repaircharacteristics of different defect geometries (Hamptonet al ., 1989; Ahlgren et al., 1994; Ethier et al., 1994).In this study, large box defects were applied with therationale that, in the case of human IVD herniation,large AF tissue defects are associated with the highest

Table 1. Applied histological criteria

Criteria De nition Staining

State of annulus defect Annulus defect was de ned as closed,when it was found completely sealed withrepair tissue and/or annulus implant in allsections analysed

Modi ed Masson s trichrome (mt),FAST, Toluidine Blue Fast Green (tb-fg)

Annulus damage

A 8 mm range of interest (ROI) adjacentto both sides of the annulus defect wasde ned for analysis

mt, FAST, tb-fg

Moderate annulus damage was de nedas few or none delaminations of annular

bres in ROI Severe annulus damage was de ned

as pronounced delamination anddisorganization of annular bres in ROI

Repair tissue thickness Three measurements of repair tissuethickness were performed in each axialsection: in the midsection of the defectand at both integration zones with theadjacent AF, respectively.

mt, FAST, tb-fg

A mean value was calculated for eachgroup at each time-point

Supra-annular scar tissue measurement of supra-annular scar tissueformation was performed at the thickestpart of scar tissue between the outer borderof repair tissue and/or implant and psoasmuscle bres, clearly identi able in Masson strichrome staining

mt

Repair tissue integration Tissue continuity of repair tissue with eachadjacent annulus brosus bre was assessedand set into proportion

mt, FAST

Toluidine Blue/Fast Green ratios Toluidine Blue and Fast Green stainingproportions were assessed within the repairtissue and/or implant

tb-fg

tb > fg ( > ), tb fg, tb < fg(< )Cell proliferation cluster Nucleus pulposus in and under the defect as

well as under the annulus brosus 8 mmadjacent to the annulus defect was de nedas ROI

mt, FAST

Cell proliferation clusters were de ned asclusters of 3 cells

FAST, Fast Green, Alcian Blue, Safranin-O, tartrazine, according to modi ed protocols of Leung et a l. (2009)




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reoperation rate of up to 21% because of reherniation(Carragee et al ., 2003).

In up to 2 weeks follow-up, the annulus implant groupshowed a clear advantage in terms of providing a mechan-ical barrier for herniating nucleus pulposus tissue incomparison to the control defect group (Figure 2d).This indicates a good primary stability in the phasepreceding stable biological integration of the implant.In this animal model, biological integration of theimplant was seen after 6 weeks follow-up (Figure 4i).For human application, in a degenerative AF environ-ment, biological integration might take considerably longer. Primary stability of the annulus implant istherefore likely an important aspect for this therapeu-tic strategy.

Box defects in canine and caprine models werereported to heal from the periphery inward with broustissue (Hampton et al., 1989; Ethier et al., 1994). Simi-larly, a consistent repair of the control defects could beobserved after 6 weeks (Figure 2d). At all follow-ups,however, a superior repair tissue thickness was shown with the annulus implant (Figure 4g). Although thestability results after 2 weeks are very clear (Figure 2d),the data from this study does not allow the conclusion whether superior repair tissue thickness translates intohigher protection against herniation incidents at latertime-points. The results, however, indicate our originalhypothesis to be correct that repair tissue thickness canbe improved by providing a biological scaffold in the innerregions of the AF, which are otherwise left unrepaired.

Figure 2. (a,b) Photomicrographs of axial sections of a control disc without annulus defect, stained with FAST and modi ed Masson s-trichrome, respectively. Annulus brosus (AF) and nucleus pulposus (NP) can be clearly identi ed. (c,e) Axial sections of 2-week spec-imens stained with FAST, with annulus box defects after pressure provocation: (c) nucleus pulposus tissue herniation (NP); (e) annu-lus implant (IMP) functioning as a barrier for nucleus pulposus tissue (NP). (d) Bar diagram showing the incidence of open and closedannulus defects as assessed by histological analysis (* p < 0.01). (f) Incidence of annulus damage, showing severe annulus damagedirectly after implantation in comparison with the control defect (* p < 0.01). At later time-points, however, no difference is evidentbetween the two groups




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After 2 weeks, a homogenous cell in ltration of theannulus implant was detected (Figure 3a,b). After 6 weeks, directional collagen bre bundles were observed(Figure 3c,d). The biomechanical forces operating on the

repair tissue probably cause the directional alignment.In this study, contrast media leakage was not able todifferentiate reliably between the different groups andtime-points. The unexpected observation that even freshannulus defects would display considerable containmentcharacteristics points to a predominant in uence of thenucleus pulposus on contrast media containment. There,macroscopically and histologically (Figure 2c), the migra-tion of nucleus pulposus into untreated annulus defects was observed, probably resulting in a tamping effect withsimilar sealing characteristics with regard to contrast me-dia as with the annulus implant. Concordantly, histologi-cal analysis in the early follow up showed highproteoglycan contents in the defect group, resulting inlater brous transformation; whereas the implant groupappears to already form more brous tissue in the early

follow-up (Figure 5c). The biomechanical implications of these ndings are, however, unclear.

In terms of primary or biological repair techniques for AFdefects, two approaches can be identi ed in the literature:

(1) suturing techniques for slit defects (Ahlgren et al.,2000; Heuer etal ., 2008, Chiang etal ., 2011) and (2) cover-ing or plugging techniques for substantial tissue defects.

Closure strategies of substantial tissue defects are of high clinical relevance. One group attempted the sealingof an AF box defect with a non-absorbable viscoelasticpolymer-based biomaterial that was injected into an ovineannulus defect (Brand, 2003). At explantation, the bioma-terial was found outside of the AF in 100% of the cases, whereas traces of the biomaterial were located only in42% in the AF defect. This example illustrates ratherdramatically that implant dislocation is one of the majorproblems in the eld and underlines the need for xationstrategies for primary implant stability. Similarly, inanother study reporting the use of absorbable collagen

glycosaminoglycan scaffolds, with and without autologous

Figure 3. Photomicrographs displaying central parts of the annulus implant in axial sections stained with modi ed Masson strichrome. (a,b) In 2-week specimens, the polyvinylidinedi uoride (PVDF) net (asterisk) and the polyglycolic acid (PGA) bres(arrow) can be clearly identi ed. In between the scaffold structures, in ltration of cells (red) and a beginning matrix assembly isobserved. (c,d) In 6-week specimens, PGA bres are completely absorbed and replaced by an increasing amount of repair tissuethat is characterized by directional bre bundles (blue). The PVDF net (asterisk) appears to be well-integrated within it. (e,f) Inthe 12-week specimens, an increased matrix density can be observed, as well as progressive attening of the cells (red)




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AF cells, plugged into box defects of a caprine model(Saad, 2007), the nucleus was often seen bulging outof the disc and a weak tissue integration of the scaffoldin the defect was reported. The additional presence of

autologous AF cells did not appear to have any effect. Addressing the problem of xation, Ahlgren et al.(2000) sutured a fascial autograft over box defects inan ovine model. No signi cant difference was foundbetween repaired and non-repaired annular strength,evaluated by pressure-volume testing. Ledet et al.(2009) opted for bone screw xation to af x a smallintestinal submucosa patch and plug to the outside of a box defect in an ovine model). This bioactive implant was evaluated 24 weeks after implantation. Accordingto the magnetic resonance imaging results, the implantappears to decelerate the degenerative process in com-parison with the control defect. Moreover, a superiormean maximum pressure before disc failure is reportedin comparison with the control defect. Unfortunately,50% of the implant group displayed considerably large

bridging osteophytes that would be worrying in a clinicalapplication.

In this study, the implantation site at the inner region of the AF proved to be advantageous in terms of implant

dislocation. No critical implant dislocation was observed. As expected, the implants bulged into the defect but neverexceeded the outer annulus border. Moreover, no pro-nounced in ammatory reactions or expansive scar tissue were observed in comparison with the control defect.However, this implantation method is demanding, andmultiple attempts for correct suture placement. Thisprocedure in icts damage to the AF adjacent to the defect(Figure 2f). This underlines the importance of a preciseapplication instrument for the implantation procedureto avoid unnecessary perforation of the adjacentannulus and to allow a practicable and safe work owin a clinical setting where the spinal canal with itsnervous structures has to be passed for implant appli-cation. Safety, precision and reproducibility of theapplication procedure, along with the integration into

Figure 4. (a,c) A 12-week specimen with well-intergrown annulus implant (IMP); (b) note the reddish muscle layer (asterisk) and thethin scar tissue layer in between the muscle layer and the repaired annulus defect. (d f) A 12-week specimen with control defect, cov-

ered with repair tissue (RT). Thus (c) and (f) display the integration zone of repair tissue with the annulus brosus (AF). (g) Box plot with whiskers minimum to maximum showing superior repair tissue thickness with the annulus implant group at all time-points. Re-pair tissue thickness in the control defect group increases from 2 to 12 weeks (* p < 0.05), but does not reach the levels of the annulusimplant group. (h) Box plot with whiskers minimum to maximum revealing no signi cant differences in the thickness of supra-annu-lar scar tissue in between groups and in chronological sequence of 12 weeks. (i) Bar diagram showing moderate integration ( < 50%)of the annulus implant in comparison with no repair tissue in the annulus defect group after 2 weeks (* p < 0.01) and post-pressureprovocation. After 12 weeks, almost complete repair tissue integration (50 100%) is achieved in both groups (# p < 0.005)




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the surgeons work ow, will be the major determinants

for acceptance in the surgical community and we expectto see a considerable evolution from the basic techniquepresented in this study. After 6 weeks, progressiveannulus damage could also be observed around thecontrol defects (Figure 2f). This is probably because of the underlying degenerative process affecting bothgroups equally in this study after applying an annulusdefect (Figure 5d f) (Johnson et al ., 2001; Guder et al .,2009). In contrast, the acute damage in icted withimplantation appear to have a moderate healing poten-tial. Thus, at later time-points, no difference of annulusdamage between both groups was seen. Therefore, this

xation method is possibly a valuable option, especially in the light of the lack of promising alternatives.In conclusion, the biointegrative annulus implant

investigated showed promising results with regard tobiointegration, enhancement of repair tissue and functionas a mechanical barrier in an ovine model.

Con ict of interest

Christian Kaps is an employee of TransTissue TechnologiesGmbH (Berlin, Germany). The other authors declare thatthey have no additional competing interests.

Acknowledgements

This study was supported by the Federal Ministry of Educa-tion and Research (BioInside 13N9831, 13N9830 and13N9827). The authors are very grateful to Prof. LotharSchilling for his technical advice, to Marijas Jurisic andMichaela Endres for their technical assistance, to ThorstenDeichmann and Annahit Arshi from the Insti tut frTextiltechnik, Faculty for Mechanical Engineering, RWTH Aachen University, for providing the annulus implant, toProf. Kirsten Schmieder for her organizational support andto Dr James Melrose for sharing some of his excellenthistological insights.

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Figure 5. (a,b) Photomicrographs of axial sections stained with Toluidine Blue Fast Green of 6- and 12-week specimens, respectively, with annulus implants, illustrating dif ferent Toluidine Blue Fast Green ratios in the repair tissue. (c) Bar diagram displaying differentToluidine Blue Fast Green ratios in the repair tissues. After 6 weeks, the control defect group shows higher proportions of ToluidineBlue staining than the annulus implant group (* p < 0.05). In contrast, after 12 weeks, the control defect group shows a considerably higher proportion of fast green staining (# p < 0.02). (d,e) Photomicrographs of axial sections stained with modi ed Masson strichrome. (d) Nucleus pulposus from a control disc without annulus defect; single cells (reddish) embedded in matrix can be ob-served. (e) Disc cell proliferation clusters (arrow) in the nucleus pulposus of a 6-week specimen from the control defect group. (f)Bar diagram showing the incidence of pronounced disc cell proliferation clusters after 6 weeks in both groups in comparison withcontrols (* p < 0.05), indicating a degenerative process


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Hegewald Et Al-2013-Journal of Tissue Engineering and Regenerative Medicine

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