JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE

RESEARCH
J Tissue Eng Regen Med (2013)
Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1831

ARTICLE

Enhancing tissue repair in annulus brosus defects

of the intervertebral disc: analysis of a bio-integrative
annulus implant in an in-vivo ovine model
Aldemar Andres Hegewald1,2*, Fabian Medved1, Daxiong Feng1,3, Charalambos Tsagogiorgas4,
Anja Beierfu5, Genevieve Ama Kyremaa Schindler1, Marcus Trunk6, Christian Kaps7,
Demissew Shenegelegn Mern1 and Claudius Thom2
1

Department of Neurosurgery, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany
Department of Neurosurgery, Innsbruck Medical University, Innsbruck, Austria
3
Spinal Surgery Department, The Afliated Hospital of Luzhou Medical College, Luzhou, Sichuan Province, P R China
4
Department of Anaesthesiology and Critical Care Medicine, University Medical Centre Mannheim, Heidelberg University,
Mannheim, Germany
5
Department of Small Animal Surgery and Ophthalmology, Ludwig Maximilians University Munich, Munich, Germany
6
Institute of Pathology, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany
7
TransTissue Technologies GmbH, Berlin, Germany
2

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 replacement 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, followed
by randomized insertion of the textile polyglycolic acid/polyvinylidene uoride annulus implant in
one of the defects. Explantation was conducted after 2, 6 and 12 weeks, followed by provocative
pressure testing and histological analysis. At 2 weeks follow-up, all specimens of the control defect
group demonstrated uncontained herniated nucleus pulposus tissue in the annulus defects. For the treated
specimens, the annulus implant consistently provided an effective barrier for herniating nucleus pulposus
tissue, with no implant dislocation at all time-points. After 2 weeks, a homogeneous cell inltration of the
annulus implant was observed, leading to a progressive directional matrix build-up. Repair tissue thickness was signicantly stronger with the annulus implant at all follow-ups (p < 0.01). No pronounced
foreign body reaction and no difference in the amount of supra-annular scar tissue over the defect sites
were observed. The implantation procedure inicted annulus damage adjacent to the defect. At later
time-points, however, no difference in comparison with the control defect group was evident. The investigated biointegrative annulus implant showed promising results with regard to biointegration, enhancement 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
*Correspondence to: Aldemar Andres Hegewald, Department of
Neurosurgery, University Medical Centre Mannheim, Heidelberg
University, Theodor-Kutzer-Ufer 13, 68167 Mannheim, Germany.
E-mail: aldemar.hegewald@medma.uni-heidelberg.de

Annulus brosus (AF) repair techniques for the

intervertebral disc (IVD) are of interest to the spine surgeon because (1) they address the unsolved problem of

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

reherniation through the untreated annulus defect

after IVD herniation (Barth et al., 2008) and (2) they
facilitate the development of nucleus pulposus replacement techniques for IVD diseases by providing
adequate 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; Intrinsic
Therapeutics, 2012). These techniques were only partly
successful and were often complicated by implant dislocation. Moreover, there is the concern of long-term effects of
solid implants on adjacent tissue structures. New generations of these implants are now in the process of controlled clinical trials.
Advances in material sciences and biotechnological
techniques are now encouraging innovative biological
repair strategies. Biological scaffold technology plays
a major role in providing primary stability and threedimensional spaces for tissue formation by introducing a whole array of absorbable and non-absorbable
biomaterials (Ratner and Bryant, 2004; Chan and
Leong, 2008). In addition, bioactive factors, enforcing
cell 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 repair
process. Cell-based approaches with multipotent progenitor
cells 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 cellfree, biointegrative annulus implant to enhance tissue
repair 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 high
bioabsorbability.
The natural repair process of AF defects advances
from the outside to the inside, but rarely exceeds the
outer third of the defect, leaving the inner regions
of the AF unrepaired (Key and Ford, 1948; Smith
and Walmsley, 1951; Hampton et al., 1989; Ethier
et al., 1994; Melrose et al., 2008). This nding and
the authors experience in biomechanical studies that
AF implants demonstrate a certain amount of bulging
under load simulation (Hegewald et al., 2009) lead to
the hypothesis of this study that an optimal implantation site for an AF implant is at the inner wall of the
AF, allowing some bulging into the AF defect without
compromising the spinal canal. At the same time, the
AF implant can exert its function as a biological scaffold by enabling repair processes in the inner regions
of the AF, otherwise left unrepaired. In this way, the
establishment of a more reliable AF repair process
with enhanced primary stability and repair tissue
strength is expected.

2. Methods
2.1. Annulus implant
A customized triphase AF implant 10 mm wide, 5 mm
high and 2 mm thick, consisting of two outer phases of
absorbable polyglycolic acid (PGA) nonwoven and a
centric phase of a non-absorbable polyvinylidene uoride
(PVDF) mesh was fabricated and assembled. The PGA
(Purac Biomaterials, Gorinchem, the Netherlands) was
melt spun to multilament bres and processed to a
non-woven, characterized by a porosity of 80% and an
interconnecting pore network, feasible for cell in-growth
and matrix build-up. The microstructure of the nonwoven consists of short laments arranged in all directions and bonded together mechanically using needle
punching. The stabilizing textile mesh is made of
non-absorbable PVDF bers with a diameter of 112 m
(G. Krahmer GmbH, Buchholz, Germany). This textile
mesh enforces primary stability, especially at the xation
points of the implant. The mesh was produced using a
double 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 outer
phases) were assembled by customized needle punching.

2.2. Surgical procedure

Twenty healthy adult merino sheep, aged 46 years,
mean body weight 82 kg, were operated on within this
project with the approval of the local competent
authority (Regierungsprsidium Karlsruhe, le number
35-9185.81/G-15/09).
The rst two sheep were operated via a dorsal interlaminar approach. In contrast to the human situs, it was
found that the outgoing spinal nerve roots are not protected
by a dural sleeve and are therefore very sensitive to mobilization. Both sheep were killed post-operatively because of
high-grade hind-limb weakness, and a left lateral retroperitoneal approach was then adopted for the study.
Two sheep developed a severe wound infection and had
to be killed prematurely. Consequently, 16 sheep were
included in the study.
After suitable intramuscular preliminary medication
with xylazine (0.10.15 mg/kg body weight) and ketamine (10 mg/kg body weight), the sheep was intubated
and a large stomach tube applied. The maintenance of
narcosis was provided by inhalation anaesthesia with
isourane (1.53 vol %) and supported by intravenous
fentanyl (13 g/kg body weight) if required. Perioperative antibiotic prophylaxis was provided with intravenous
cefazolin (2 g). Intraoperative monitoring was established
with arterial blood pressure measurement and measurement of oxygen saturation.
The sheep was carefully bedded on the right side. Using
standard sterile techniques, the lumbar segments L3/4
and L4/5 were accessed via a left retroperitoneal, transpsoas

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

Enhancing biological annulus repair

(between psoas major and minor) approach. After uoroscopic examination for level localization, two box-shaped
annulus defects of 3.5 3.5 mm were applied with a scalpel
to the two intervertebral discs. Protruding nucleus pulposus
tissue in the defect was carefully coagulated and the adjacent
AF was undermined with a hook instrument. The nucleus
pulposus was otherwise left intact and no nucleotomy
was performed. A blocked randomization for the annulus implant insertion was then conducted.
Directly before application, the annulus implant was
immersed in autologous blood serum for potential
bioactivation (Hegewald et al., 2011). The implantation
was achieved with an inside-out xation technique, using
non-absorbable sutures (4-0 Prolene, ETHICON GmbH,
Norderstedt, Germany) that were prexed at the four corners of the annulus implant (Figure 1). With this technique,
the annulus implant could be pulled inside the disc and
tightly xed to the inner AF.

2.3. Explantation procedure and provocative

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

annulus defect with implant and (c) intact control. In the

analysis, (a) and (b) are referred to as time-point 0 weeks.
Following this procedure, provocative pressure testing
was performed. Under uoroscopic observation, a customized high-pressure needle was placed centrally into
the nucleus pulposus. With an angiography pump, continuous injection (0.5ml/min) of contrast medium was
applied to the nucleus pulposus compartment until contrast media leakage was observed uoroscopically. A
pressure transducer was used to continuously register
the pressure values.

2.4. Histology
Tissue processing and staining protocols were mostly based
on procedures established by Melrose et al. (2004, 2007).
Directly after provocative pressure testing, individual
IVDs were excised with a band saw and xed in 10%
neutral buffered formalin (1 week). Decalcication was
performed in several changes (three times a week) of
10% formic acid in 5% neutral buffered formalin with
constant agitation (4 weeks). After decalcication, the
specimens were rinsed in running tap water (30 min)
and equilibrated in several changes of 70% ethanol.
To prepare tissue sections, specimens were doubleembedded in parafncelloidin. Specimens were rst
dehydrated 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. (df) Demonstration
of the intraoperative implantation procedure via a retroperitoneal approach: (d) application of an annulus box defect; (e) pulling the
annulus implant inside the disc with preset sutures; (f) completed implantation and adjacent control defect
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

A. A. Hegewald et al.

equilibrated in methyl-benzoate for 24 h. To inltrate

the tissue with celloidin, specimens were incubated in
a mixture of celloidin and methylbenzoate. First, they
were equilibrated in methylbenzoate containing 1%
celloidin, then subsequently in methylbenzoate containing
5% celloidin. After purication with chloroform for 1 hour,
the specimens were vacuum-inltrated and embedded in
Paraplast (Paraplast Plus, Sigma-Aldrich, Germany). Before cutting, the surface was softened by using softening solution containing ammonia, glycerine, 100% ethanol and
distilled water. Afterwards, 10 m sections were cut with
a microtome and xed on star frost plus (SuperFrost Plus
Objekttrger, R. Langenbrinck Labor- und Medizintechnik,
Emmendingen, Germany) glass slides. Sections were dried
at 75C on a heating plate (10 min) and then overnight in
an oven at 55C. Deparafnization was performed in xylene.
Thereafter, specimens were rehydrated with graded ethanol
washes (10070% v/v).
The following stains were performed: (1) modied
Massons trichrome, (2) toluidine bluefast green, both
according to modied protocols established by Melrose
et al. (2007), (3) FAST (Fast Green, Alcian Blue,
Safranin-O, tartrazine) according to modied protocols
of Leung et al. (2009). For the Massons trichrome
staining, the sections were rst incubated in Bouins
mordant xative at 60C (1 h) and afterwards puried
in 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 puried
in running tap water and Scotts tap water substitute
(3 min) and stained in Ponceau de Xylidine (8 min).
After being washed with distilled water, sections were
differentiated in 5% phosphotungstic acid solution
(10 min) and counterstained with 1% Fast Green
FCF (2 min). Finally, sections were dehydrated in several
changes of absolute ethanol and cleared in xylene. With
Massons trichrome, cytoplasm and muscle bres stained
red and collagen bres stained blue. Somewhat unexpected
was the consistent reddish staining of the AF; this was
also found in the repair tissue of all 12-weeks specimens.
For Toluidine BlueFast Green staining, sections were
stained in 0.04% Toluidine Blue O (10 min) and then
counterstained with 0,1% Fast Green FCF (2 min),
puried with several changes of isopropanol and cleared
in xylene. With Toluidine BlueFast Green staining,
proteoglycans are stained blue. For multichromatic
FAST (Fast Green, Alcian Blue, Safranin-O, tartrazine)
staining, initial staining was performed with Alcian
Blue 1% (2.5 min) and Safranin-O 0.1% (3 min). After
differentiation with ethanol 50% (1 min) counterstaining
was performed with tartrazine 0.25% (10 s) and Fast Green
0.001% (5 min). Finally, sections were dehydrated in
absolute ethanol and cleared in xylene. With FAST
staining, acidic glycoproteins stained blue, neutral
glycoproteins appeared orange and brous tissue
appeared yellowish. After each staining and differentiation step, sections were briey rinsed in distilled
water and mounted in eukitt (EUKITT, O. Kindler
GmbH, Freiburg, Germany) after the staining procedure was completed.

2.5. Histological analysis

Histological analysis was performed with assistance of the
digital image processing software for microscopes Axiovision
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 (SD
3.7) 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 version
5.0d for Mac OS X (GraphPad Software, San Diego, CA,
USA). Quantitative data from both groups at single timepoints were compared using the nonparametric Mann
Whitney test (two-tailed). For data comparison at different time-points, the non-parametric KruskalWallis test
with Dunns post test was applied. Categorical variables
were analysed with Fishers exact test (two-sided). The
signicance level for all the statistical tests was p = 0.05.

3. Results
Sixteen sheep were included in the study. They did not show
any anaesthesia or surgically related complications. There
were no device-related or other health-related complications.
Contrast media leakage with provocative pressure
testing directly after explantation was observed at a mean
pressure of 0.53 MPa, with high variance within the
groups. There were no statistical signicant differences
between groups after 2, 6 and 12 weeks. Similarly, there
were no differences within groups at different timepoints. The intact control did not show any contrast media
leakage up to the pressure limit of 4.8 MPa of the system.
At the time-points 0 weeks and 2 weeks, all 10 specimens
of the control defect group demonstrated uncontained
herniated nucleus pulposus tissue in the annulus defects
(Figure 2c,d). For the treated specimens, the annulus
implant consistently provided an effective barrier for herniating nucleus pulposus tissue (Figure 2d,e) with no implant
dislocation at all time-points. After 6 weeks and 12 weeks,
both groups showed comparable results with regard to
closing the annulus defect.
The implantation procedure inicts annulus damage
adjacent to the defect in comparison to sole injury of the
annulus (Figure 2f). At later time-points, however, no
differences were evident.
After 2 weeks, a homogeneous cell inltration, including
the central parts of the annulus implant, is observed
(Figure 3a,b). After 6 weeks, the absorbable component
(PGA) of the annulus implant completely degraded
and degradation products could only sporadically be
found in some specimens (Figure 3c,d). The remaining
non-absorbable component (PVDF) was found to be
steeped by repair tissue that impressed with directional

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

Enhancing biological annulus repair

Table 1. Applied histological criteria
Criteria
State of annulus defect

Annulus damage

Repair tissue thickness

Supra-annular scar tissue

Repair tissue integration

Toluidine Blue/Fast Green ratios

Cell proliferation cluster

Denition

Staining

Annulus defect was dened as closed,

when it was found completely sealed with
repair tissue and/or annulus implant in all
sections analysed
A 8 mm range of interest (ROI) adjacent
to both sides of the annulus defect was
dened for analysis
Moderate annulus damage was dened
as few or none delaminations of annular
bres in ROI
Severe annulus damage was dened
as pronounced delamination and
disorganization of annular bres in ROI
Three measurements of repair tissue
thickness were performed in each axial
section: in the midsection of the defect
and at both integration zones with the
adjacent AF, respectively.
A mean value was calculated for each
group at each time-point
measurement of supra-annular scar tissue
formation was performed at the thickest
part of scar tissue between the outer border
of repair tissue and/or implant and psoas
muscle bres, clearly identiable in Massons
trichrome staining
Tissue continuity of repair tissue with each
adjacent annulus brosus bre was assessed
and set into proportion
Toluidine Blue and Fast Green staining
proportions were assessed within the repair
tissue and/or implant
tb > fg (> ), tb fg, tb < fg(< )
Nucleus pulposus in and under the defect as
well as under the annulus brosus 8 mm
adjacent to the annulus defect was dened
as ROI
Cell proliferation clusters were dened as
clusters of 3 cells

Modied Massons trichrome (mt),

FAST, Toluidine BlueFast Green (tb-fg)
mt, FAST, tb-fg

mt, FAST, tb-fg

mt

mt, FAST
tb-fg

mt, FAST

FAST, Fast Green, Alcian Blue, Safranin-O, tartrazine, according to modied protocols of Leung et al. (2009)

bre bundles. After 12 weeks, the density of the extracellular matrix considerably increased and a progressive
attening of the cells could be observed (Figure 3e,f).
Repair tissue thickness was signicantly greater in the
annulus implant group at all follow-ups (Figure 4a,b,d,e,g).
After 2 weeks, the annulus implant group demonstrated
a moderate biological integration with the host tissue
(Figure 4i). With increasing follow-up, the stability and
amount of repair tissue integration improved in both
groups (Figure 4c,f,i). No pronounced foreign body reaction or granuloma formation was observed. Repair tissue
in both groups showed similar appearances, although
the repair tissue in the defect group appeared to be
more highly vascularized in most specimens (Figure 4f).
There was no difference in the amount of supra-annular
scar tissue over the defect site between the two groups
(Figure 4h).
At 6 weeks follow-up, the control defect group
displayed higher proportions of Toluidine Blue staining
that were site-concordant with bluish staining in FAST,
indicating high proteoglycan proportions in the repair
tissue (Figure 5c). In contrast, at later follow-up, Fast
Green staining dominated the repair tissue in both groups

(Figure 5ac), which was site-concordant with yelloworange staining in FAST, indicating a more brous
tissue with lower proteoglycan concentrations.
The 6-week follow-up was accompanied by the pronounced appearance of disc cell proliferation clusters in
the nucleus pulposus of both groups (Figure 5df), indicating 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 have
already described the formation of a brous cap in the
outer regions of the defects, leaving the inner regions
unrepaired (Key and Ford, 1948; Smith and Walmsley,
1951). Further studies reported on the specic repair
characteristics of different defect geometries (Hampton
et al., 1989; Ahlgren et al., 1994; Ethier et al., 1994).
In this study, large box defects were applied with the
rationale that, in the case of human IVD herniation,
large AF tissue defects are associated with the highest

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

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DOI: 10.1002/term

A. A. Hegewald et al.

Figure 2. (a,b) Photomicrographs of axial sections of a control disc without annulus defect, stained with FAST and modied Massonstrichrome, respectively. Annulus brosus (AF) and nucleus pulposus (NP) can be clearly identied. (c,e) Axial sections of 2-week specimens stained with FAST, with annulus box defects after pressure provocation: (c) nucleus pulposus tissue herniation (NP); (e) annulus implant (IMP) functioning as a barrier for nucleus pulposus tissue (NP). (d) Bar diagram showing the incidence of open and closed
annulus defects as assessed by histological analysis (*p < 0.01). (f) Incidence of annulus damage, showing severe annulus damage
directly after implantation in comparison with the control defect (*p < 0.01). At later time-points, however, no difference is evident
between the two groups

reoperation rate of up to 21% because of reherniation

(Carragee et al., 2003).
In up to 2 weeks follow-up, the annulus implant group
showed a clear advantage in terms of providing a mechanical barrier for herniating nucleus pulposus tissue in
comparison to the control defect group (Figure 2d).
This indicates a good primary stability in the phase
preceding stable biological integration of the implant.
In this animal model, biological integration of the
implant was seen after 6 weeks follow-up (Figure 4i).
For human application, in a degenerative AF environment, biological integration might take considerably
longer. Primary stability of the annulus implant is
therefore likely an important aspect for this therapeutic strategy.

Box defects in canine and caprine models were

reported to heal from the periphery inward with brous
tissue (Hampton et al., 1989; Ethier et al., 1994). Similarly, a consistent repair of the control defects could be
observed after 6 weeks (Figure 2d). At all follow-ups,
however, a superior repair tissue thickness was shown
with the annulus implant (Figure 4g). Although the
stability 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 into
higher protection against herniation incidents at later
time-points. The results, however, indicate our original
hypothesis to be correct that repair tissue thickness can
be improved by providing a biological scaffold in the inner
regions of the AF, which are otherwise left unrepaired.

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

Enhancing biological annulus repair

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

After 2 weeks, a homogenous cell inltration of the

annulus 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 to
differentiate reliably between the different groups and
time-points. The unexpected observation that even fresh
annulus defects would display considerable containment
characteristics points to a predominant inuence of the
nucleus pulposus on contrast media containment. There,
macroscopically and histologically (Figure 2c), the migration of nucleus pulposus into untreated annulus defects
was observed, probably resulting in a tamping effect with
similar sealing characteristics with regard to contrast media as with the annulus implant. Concordantly, histological analysis in the early follow up showed high
proteoglycan contents in the defect group, resulting in
later brous transformation; whereas the implant group
appears 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 AF
defects, two approaches can be identied in the literature:
(1) suturing techniques for slit defects (Ahlgren et al.,
2000; Heuer et al., 2008, Chiang et al., 2011) and (2) covering or plugging techniques for substantial tissue defects.
Closure strategies of substantial tissue defects are of
high clinical relevance. One group attempted the sealing
of an AF box defect with a non-absorbable viscoelastic
polymer-based biomaterial that was injected into an ovine
annulus defect (Brand, 2003). At explantation, the biomaterial was found outside of the AF in 100% of the cases,
whereas traces of the biomaterial were located only in
42% in the AF defect. This example illustrates rather
dramatically that implant dislocation is one of the major
problems in the eld and underlines the need for xation
strategies for primary implant stability. Similarly, in
another study reporting the use of absorbable collagen
glycosaminoglycan scaffolds, with and without autologous

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A. A. Hegewald et al.

Figure 4. (a,c) A 12-week specimen with well-intergrown annulus implant (IMP); (b) note the reddish muscle layer (asterisk) and the
thin scar tissue layer in between the muscle layer and the repaired annulus defect. (df) A 12-week specimen with control defect, covered 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. Repair tissue thickness in the control defect group increases from 2 to 12 weeks (*p < 0.05), but does not reach the levels of the annulus
implant group. (h) Box plot with whiskers minimum to maximum revealing no signicant differences in the thickness of supra-annular 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-pressure
provocation. After 12 weeks, almost complete repair tissue integration (50100%) is achieved in both groups (#p < 0.005)

AF cells, plugged into box defects of a caprine model

(Saad, 2007), the nucleus was often seen bulging out
of the disc and a weak tissue integration of the scaffold
in 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 in
an ovine model. No signicant difference was found
between repaired and non-repaired annular strength,
evaluated by pressure-volume testing. Ledet et al.
(2009) opted for bone screw xation to afx a small
intestinal submucosa patch and plug to the outside of
a box defect in an ovine model). This bioactive implant
was evaluated 24 weeks after implantation. According
to the magnetic resonance imaging results, the implant
appears to decelerate the degenerative process in comparison with the control defect. Moreover, a superior
mean maximum pressure before disc failure is reported
in comparison with the control defect. Unfortunately,
50% of the implant group displayed considerably large

bridging osteophytes that would be worrying in a clinical

application.
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 never
exceeded the outer annulus border. Moreover, no pronounced inammatory reactions or expansive scar tissue
were observed in comparison with the control defect.
However, this implantation method is demanding, and
multiple attempts for correct suture placement. This
procedure inicts damage to the AF adjacent to the defect
(Figure 2f). This underlines the importance of a precise
application instrument for the implantation procedure
to avoid unnecessary perforation of the adjacent
annulus and to allow a practicable and safe workow
in a clinical setting where the spinal canal with its
nervous structures has to be passed for implant application. Safety, precision and reproducibility of the
application procedure, along with the integration into

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

Enhancing biological annulus repair

Figure 5. (a,b) Photomicrographs of axial sections stained with Toluidine BlueFast Green of 6- and 12-week specimens, respectively,
with annulus implants, illustrating different Toluidine BlueFast Green ratios in the repair tissue. (c) Bar diagram displaying different
Toluidine BlueFast Green ratios in the repair tissues. After 6 weeks, the control defect group shows higher proportions of Toluidine
Blue 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 modied Massons
trichrome. (d) Nucleus pulposus from a control disc without annulus defect; single cells (reddish) embedded in matrix can be observed. (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 with
controls (*p < 0.05), indicating a degenerative process

the surgeons workow, will be the major determinants

for acceptance in the surgical community and we expect
to see a considerable evolution from the basic technique
presented in this study. After 6 weeks, progressive
annulus damage could also be observed around the
control defects (Figure 2f). This is probably because of
the underlying degenerative process affecting both
groups equally in this study after applying an annulus
defect (Figure 5df) (Johnson et al., 2001; Guder et al.,
2009). In contrast, the acute damage inicted with
implantation appear to have a moderate healing potential. Thus, at later time-points, no difference of annulus
damage 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 to
biointegration, enhancement of repair tissue and function
as a mechanical barrier in an ovine model.

Conict of interest
Christian Kaps is an employee of TransTissue Technologies
GmbH (Berlin, Germany). The other authors declare that
they have no additional competing interests.

Acknowledgements
This study was supported by the Federal Ministry of Education and Research (BioInside 13N9831, 13N9830 and
13N9827). The authors are very grateful to Prof. Lothar
Schilling for his technical advice, to Marijas Jurisic and
Michaela Endres for their technical assistance, to Thorsten
Deichmann and Annahit Arshi from the Institut fr
Textiltechnik, Faculty for Mechanical Engineering, RWTH
Aachen University, for providing the annulus implant, to
Prof. Kirsten Schmieder for her organizational support and
to Dr James Melrose for sharing some of his excellent
histological insights.

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

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DOI: 10.1002/term