Tendon bone healing can be enhanced by demineralized bone matrix: A functional and histological study

Tendon Bone Healing Can Be Enhanced by DemineralizedBone Matrix: A Functional and Histological Study

Siva Sundar, Catherine J. Pendegrass, Gordon W. Blunn

Centre for Biomedical Engineering, Institute of Orthopaedics and Musculoskeletal Science, University College London,Brockley Hill, Stanmore, Middlesex, HA7 4LP, UK

Received 31 May 2007; revised 7 January 2008; accepted 21 April 2008Published online 5 August 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31157

Abstract: Rotator cuff repair surgery has high failure rates, with tendon reattachment to

bone remaining a challenging clinical problem. Increasing the integrity of the healing tendon-

bone interface has been attempted by adopting a number of different augmentation strategies.

Because of chondrogenic and osteogenic properties we hypothesise that demineralized bone

matrix (DBM) augmentation of a healing tendon-bone interface will result in improved

function, and a morphology that more closely resembles that of a normal enthesis, compared

with nonaugmented controls in an ovine patellar tendon model. The right patellar tendon was

detached from its insertion and reattached to an osteotomized bone bed using suture anchors.

Two groups were analyzed, the control group (without augmentation) and the DBM group

(DBM interposed between the tendon and bone). Animals were sacrificed at 12 weeks. Force

plate, mechanical, and histomorphometric analyses were performed. Tendon repairs failed at

a rate of 33 and 0% for the control and DBM groups, respectively. DBM augmentation

resulted in significantly improved functional weight bearing and increased amounts of

fibrocartilage and mineralized fibrocartilage. This study shows that DBM enhances tendon-

bone healing and may reduce the high failure rates associated with rotator cuff repair

clinically. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 88B: 115–122, 2009

Keywords: demineralized bone matrix; tendon to bone healing; rotator cuff repair

INTRODUCTION

Tendon reattachment to bone following rotator cuff repair

is a challenging clinical problem. Rotator cuff tears are

common, and studies have shown that the prevalence of

partial and thickness tears is �18.45 and 11.75%, respec-

tively.1 Despite these high incidence rates, rotator cuff

repair is not associated with clinically good outcomes, with

failure rates up to 90% for massive tear repairs being

reported.2 The most commonly reported failure rates are

between 20 and 65%,3 most of which occur in the early

stages of healing. Following re-repair, 83% of patients

report fair or poor outcomes4 predominantly due to tendon

avulsion from bone at the repair site.5

One of the principal reasons for these failures is pro-

longed immobilization of the tendon, brought about by

weakness and stiffness occurring as a consequence of

decreasing total collagen content.6 Although the tendon

repair requires protection from early mobilization to pre-

vent failure, early mobilization is considered beneficial for

biological healing at the tendon–bone interface. As a result

of this paradox, most tendon reattachment research has

focused on improving initial fixation strength to allow for

early mobilization,7–9 whilst maintaining the integrity of

the tendon repair until biological healing has occurred.

Recently, attempts have been made to enhance healing

at the tendon–bone interface. Rodeo et al.10 postulated that

increasing bone growth at the interface would improve ten-

don–bone healing and growth factor, cell-based strategies

and scaffolds have been used to augment healing. Studies

using growth factors, such as BMPs, have been shown to

be successful in improving pull-out strengths of repairs11

and mesenchymal stem cell-based strategies have shown

promising results in ACL reconstruction in a rabbit model12

exhibiting a fibrocartilaginous interface at earlier time-

points than nontreated animals. In contrast, xenogenic small

intestine submucosa has shown disappointing results in

clinical trials.13 To date, none of these approaches have

become routine clinical practice.

Demineralized bone matrix (DBM) is known to be

osteoinductive via endochondral ossification14,15 and is a

slow release system for bone morphogenetic proteins

(BMPs).16 In 1965 Urist speculated about the existence of

BMPs following research into DBM and its ability to

induce bone formation in ectopic sites in vivo.14 DBM is

currently used clinically in fracture nonunion17 and in

Correspondence to: Dr. C. J. Pendegrass (e-mail: [email protected])

' 2008 Wiley Periodicals, Inc.

115

spinal fusion18 to augment bone formation and has been

shown to transdifferentiate fibroblasts into chondrocytes

in vitro.19

Tendon–bone healing occurs through progressive miner-

alization of tendon through bone growth and subsequent

remodeling of tissues at the interface under mechanical

strain.20 DBM is osseoinductive by endochondral ossifica-

tion14 and its ability to act as a suitable scaffold for many

cell types, make it a potential scaffold to augment tendon–

bone healing where a transition from bone, to mineralized

cartilage, nonmineralized cartilage through to tendon is

required. We postulate that DBM might be suitable to aug-

ment tendon–bone healing in an in-vivo model. The ovine

patellar tendon model is a more discerning model of tendon

reattachment than the rotator cuff as there are no other

compensatory structures and the extensor mechanism of the

lower leg depends on it. This model enables force plate

analysis to be used as an outcome measure of the integrity

of the repair.21

We hypothesize that DBM augmentation of a healing

tendon–bone interface will result in improved function and

a morphology that more closely resembles that of a normal

enthesis, compared with nonaugmented controls in an ovine

patellar tendon model.

MATERIALS AND METHODS

Study Design

Our established ovine patellar tendon model was used,22,23

in accordance with a Project License protocol accepted

under the UK Home Office Animals (Scientific Procedures)

Act 1986. In all 19 skeletally-mature ovine Friesland ewes

(64–94 kg) were randomly allocated to either the control

group (n 5 11) or the DBM group (n 5 8). In the control

group, tendon–bone reconstruction was achieved using 3

Mitek Panalok RC QuickAnchor Plus(suture anchors

(Ethicon, Somerville, NJ). In the DBM group, reconstruc-

tion was augmented with a piece of allogeneic DBM which

was interposed between the tendon and bone. Animals

were force plate analyzed preoperatively and at 3-, 6-, 9-,

and 12-week postoperation. After 6 weeks the tibial–patel-

lar tendon enthesis from two animals in each group were

processed for qualitative histology. Additionally, the tibial–

patellar tendon construct from six animals, three in each

group, was tested to failure. Histology on the enthesis in

the remaining animals was carried out at 12 weeks.

DBM Manufacture

DBM was manufactured according to Urist’s protocol.14

The infraspinatus fossae from the scapulae of one female

sheep were removed and stripped of periosteum. Strips

were between 2 and 3 mm in thickness and were placed in

0.6N HCl for 8 h. Complete demineralization was con-

firmed by radiographic analysis (Raymax, Elstree, Middle-

sex, UK). This was followed by prolonged washing in

0.15M NaCl and lyophilisation (BOC Edwards, Crawley,

West Sussex, UK) for storage. Strips measuring 15 330 mm2 were cut and sterilized by gamma irradiation at a

dose of 25 KGreys (Isotron, Reading, UK). Samples were

rehydrated at the time of surgery in normal saline for

45 min prior to use.

Force Plate Analysis

Animals underwent force plate analysis preoperatively and

3-, 6-, 9-, and 12-week postoperation. Twelve readings of

each hind limb were taken by walking the animals over a

force plate (Kistler Biomechanics Limited, Alton, UK) in a

gait analysis laboratory. The mean peak vertical component

of the ground reaction force (GRFz) of each hind limb was

obtained and normalized for weight (Fmax/weight). Func-

tional weight bearing (FWB) was expressed as the mean

GRFz of the right hind limb as a percentage of the left.

Inclusion criteria included preoperation FWB within the

range of 100% 6 2%. Improvements in FWB data were

compared statistically between groups at each time point.

Surgical Procedure

Animals were anaesthetized and under aseptic conditions a

midline incision was made over the right stifle joint. The

patellar tendon was isolated [Figure 1(b)] and detached by

dissection from the tibial tuberosity [Figure 1(c)]. The tibial

tubercle was osteotomized and 3 Mitek anchors (Ethicon,

Somerville, NJ) were inserted into the flat bone bed [Fig-

ure 1(d)]. In the control group, the tendon was reattached

directly to the bone bed using the Mitek anchors [Fig-

ure 1(e)]. In the DBM group, a piece of DBM was inter-

posed between the tendon and bone prior to reattachment

of the tendon by threading the Mitek suture through the flat

DBM [Figure 1(d)]. Animals received analgesics and anti-

biotics for a maximum of 4 days after the operation. Ani-

mals were allowed to mobilize freely postoperation and

were group-housed in an indoor pen (1.3 3 2.6 m2). Ani-

mals were euthanazed at 6 and 12 weeks.

Radiographic Assessment

Radiographs of all operated limbs were taken at 3, 6, 9,

and 12 weeks. The procedure was considered to have failed

if, from the radiographs, there was recognizable Patellar

Alta indicating failure at the patellar–tibial bone interface.

Mechanical Testing

Right stifle joint tibia–patellar tendon–patella complexes (n5 6) were harvested at time zero and at 12-week postoper-

ation. These were mounted to a custom made jig on a

materials testing machine (Zwick/Roell Z005; Zwick

GmbH, Ulm, Germany). The stifle joint construct was fixed

at the patella and proximal tibia with 2-mm diameter surgi-

cal K-wires (Synthes, Stratec, Welwyn Garden City, UK),

116 SUNDAR, PENDEGRASS, AND BLUNN

Journal of Biomedical Materials Research Part B: Applied Biomaterials

Figure 2. (a) Box and whiskers plot showing percentage functional weight bearing of the DBM group

at 3 weeks. (b) Box and whiskers plot showing percentage functional weight bearing of the DBM and

control groups at 6 weeks. (c) Box and whiskers plot showing percentage functional weight bearing ofthe DBM and control groups at 9 weeks. (d) Box and whiskers plot showing percentage functional

weight bearing of the DBM and control groups at 12 weeks. [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]

Figure 1. (a) Summary schematic of control group surgery. (b) Summary schematic of DBM group sur-

gery. (c) Photograph showing isolation of the patellar tendon during surgery. (d) Photograph showingsharp dissection of the patellar tendon from the tibial tuberosity and insertion of suture anchors during

surgery. (e) Photograph showing positioning of DBM in the DBM group during surgery. (f) Photograph

showing suture anchor sutures knotted and tied down during surgery. [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]

and low melting point aluminium alloy (MCP70) (MCP

Mining and Chemical Company, Wellingborough, UK),

respectively. The complexes were tested under tension,

without preconditioning, at a displacement of 200 mm/min

in a vertical direction to obtain ultimate tensile strength

(UTS). Data were statistically compared between groups.

Histological Analysis

Samples were fixed in 10% formal saline and underwent

ascending graded alcohol dehydration, defatting in chloro-

form, and embedding in LR White Hard Grade Resin

(London Resin Company Limited, Reading, UK). Sections

were cut, ground, and polished to 70–100 lm before stain-

ing with Toluidine Blue and Paragon. Samples underwent

qualitative and quantitative morphological analysis using a

light microscope (Zeiss, Hamburg, Germany) linked to

image analysis software (Axiovision, Zeiss, Hamburg, Ger-

many). Qualitative and quantitative histology were per-

formed at 6 weeks (n 5 2) and 12 weeks (n 5 6) using

two sections from one-quarter width intervals across the

insertion sites from each animal. By measuring the length

of the tissue at the interface the percentage of the tendon–

bone interface length which was fibrous or fibrocartilagi-

nous was determined. Fibrous insertions were characterized

by the appearance of Sharpey’s-like fibres extending from

the tendon and penetrating directly into bone, and an ab-

sence of fibrocartilage.

Fibrocartilaginous insertions were characterized by the

presence of fibrocartilage with chondrocytes in lacunae

interposed between the tendon and bone. Areas of fibrocar-

tilage, mineralized fibrocartilage, and new bone were meas-

ured using image analysis software (Axiovision, Zeiss,

Hamburg, Germany).

Statistical Analysis

Numerical data were inputted in to SPSS v11 for Windows

(SPSS, Chicago, IL). Data are expressed as median values

with 95% confidence intervals. Mann Whitney U tests were

used to compare data between groups, whilst Wilcoxon

Signed Rank tests were used to assess differences within

each group over time. A Fisher’s exact test was used to

assess failure rates between the groups. Results were con-

sidered significant at the 0.05 level.

RESULTS

Failure Rate

Failures were initially detected by observation of an abnor-

mal gait and confirmed by patella alta on radiographs. Two

out of eight control animals failed within 6-week postoper-

ation due to tendon avulsion from bone. A replacement ani-

mal failed within the same time period, and further

replacements were considered unnecessary. In total, three

out of nine animals in the control group failed, correlating

to a 33% failure due to pull-out. No failures were observed

in the DBM group. Fisher’s exact test showed no signifi-

cant difference between the two groups (p 5 0.18).

Force Plate Analysis

It was considered unethical to force plate the animals in

the control group at 3 weeks due to lameness, hence no

data is presented. In the DBM group FWB reached a me-

dian of 56.1% (42.7–66.1) at 3 weeks [Figure 2(a)]. The

control and DBM groups reached median FWB of 65.5%

(57.7–70.0) and 73.5% (65.5–81.1) at 6 weeks [Figure

2(b)], 72.6% (63.9–81.1) and 89.7% (81.2–96.5) at 9 weeks

[Figure 2(c)], and 80.3% (69.7–89.1) and 94.7% (85.7–

99.4) at 12 weeks [Figure 2(d)], respectively. At 6, 9, and

12 weeks, FWB was significantly greater in the DBM

group compared with the control group (p 5 0.028, 0.004,

and 0.022, respectively). FWB in the DBM group improved

significantly between 3 and 6 weeks (p 5 0.018), and 6

and 9 weeks (p 5 0.028). The control group improved

significantly between 6 and 9 weeks (p 5 0.043) and 9 and

12 weeks (p 5 0.028).

Figure 3. (a) Photomicrograph showing appearance at 6 weeks of the control group tendon midsub-

stance (T) showing poor organization, plump rounded fibroblast nuclei, and an absence of a character-

istic crimp pattern. Specimen stained with Toluidine Blue and Paragon. Bar 5 100 lm. (b) Appearanceat 6 week of the DBM group tendon midsubstance (T) showing well organized, crimped collagen fibres

with elongated fibroblast nuclei. Specimen stained with Toluidine Blue and Paragon. Bar 5 100 lm. (c)

The control group tendon–bone interface at 6 weeks showing disorganized tendon (T), small regions of

fibrocartilage (FC) and new bone (NB). Specimen stained with Toluidine Blue and Paragon. Bar 5200 lm. (d) The 6 week DBM group tendon–bone interface showing tendon (T) comprising of organ-

ized collagen fibres, fibrocartilage (FC), small amounts of mineralized fibrocartilage (MFC), and new

bone (NB). Specimens stained with Toluidine Blue and Paragon. Bar 5 200 lm. [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4. (a) Photomicrograph showing appearance of control group tendon midsubstance (T) at12 weeks. Bar 5 100 lm. (b) 12 week DBM tendon midsubstance (T) showing organized collagen

fibres with characteristic crimp pattern. Bar 5 100 lm. (c) Appearance of control group tendon–bone

interface at 12 weeks showing tendon (T), fibrocartilage (FC), mineralised fibrocartilage (MFC), andnew bone (NB). Bar 5 200 lm. (d) 12 week DBM tendon–bone interface showing mineralized fibrocar-

tilage (MFC), and large amounts of fibrocartilage (FC). Bar 5 200 lm. [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]



Figure 3

Figure 4

Mechanical Testing

At 12 weeks all samples failed in the midsubstance and

no significant difference in UTS was observed between

the groups (p 5 0.343). The median UTS values (with

95% confidence intervals) for the control and DBM

groups were 1700.5N (1292.1–2119.6) and 2119.3N(1253.5–2814.8), respectively. The UTS where the ten-

don had been reattached in the laboratory using Mitek

anchors in cadaveric samples was 114.5N (94.7–138.6).

There was a significant difference observed between the

cadaveric samples and the DBM and control group sam-

ples at 12 weeks (p 5 0.034 and p 5 0.034, respec-

tively). In the cadaveric samples the tendon construct

failed at the insertion point and indicates that at 12 weeks

the insertion site had functionally healed in both control

and DBM groups.

Qualitative Histology

At 6 weeks, there were marked differences in morphology

between the two groups. In the control group specimens,

the tendon midsubstance was disorganized with randomly

arranged collagen fibres and rounded fibroblastic nuclei

[Figure 3(a)]. The tendon–bone interface was predomi-

nantly fibrous with small interpositional regions of fibrocar-

tilage, no mineralized fibrocartilage, and relatively small

amounts of new bone [Figure 3(b)]. In the DBM group, the

interpositional DBM had been completely remodelled. The

tendon midsubstance appeared normal, with orientated

crimped collagen fibres running in the longitudinal direc-

tion of the tendon. These fibres were interspersed with

elongated fibroblast nuclei [Figure 3(c)]. The tendon–bone

interface was predominately fibrocartilaginous with small

amounts of mineralized fibrocartilage. Compared with 6-

week control specimens there were larger regions of new

bone and a developing tidemark [Figure 3(d)].

At 12 weeks in the control group specimens, the tendon

midsubstance remained disorganized [Figure 4(a)]. The ten-

don–bone interface appeared more mature, with more

extensive fibrocartilaginous regions interposed between

zones of perforating collagen fibres extending from the ten-

don to the underlying bone [Figure 4(b)]. In this group,

mineralized fibrocartilage or new bone was rarely observed.

There was no evidence of a tidemark. In the DBM group

specimens the tendon midsubstance appeared normal

[Figure 4(c)]. Large, distinct areas of organized fibrocarti-

lage were observed with chondrocytes in their lacunae ori-

entated in the direction of the collagen fibres. Mineralized

fibrocartilage consisted of chondrocytes surrounded by min-

eralized matrix, which extended in to the fibrocartilaginous

layer above [Figure 4(d)].

Quantitative Histology

At 12 weeks, the DBM group tendon–bone interface was

significantly more fibrocartilaginous (p 5 0.025) and the

control group was significantly more fibrous (p 5 0.025).

The DBM group produced 930.0 cm2 (717.42–1404.77),

427.61 cm2 (85.2–2349.6), and 61.0 m2 of fibrocartilage,

mineralized fibrocartilage, and new bone, respectively. The

control group produced 183.63 cm2 (58.50–237.87),

7.38 cm2 (2.67–19.05), and 25.29 cm2, respectively.

The DBM group produced significantly more fibrocar-

tilage (p 5 0.025) and mineralized fibrocartilage (p 50.025), however no significant difference was observed

in the amount of new bone between the groups (p 50.655).

DISCUSSION

The aim of this study was to determine the role of DBM in

augmenting healing tendon–bone interfaces. DBM was

found to produce significantly better functional results at 6,

9, and 12 weeks and significantly more fibrocartilage and

mineralized fibrocartilage at 12-week postoperation. Walsh

et al. have performed a series of studies in which suture

anchors were used for patellar tendon reconstruction in an

ovine model.24–26 They have not yet reported any failures

in their long term studies, although this may be due to dif-

ferences in their intra and postoperative protocols from

ours. They used a whipstitch suture method to attach the

tendon to bone which may dissappate stress more than

standard surgical knots as used in this study. They also

used a modified Robert Jones bandage for 3 weeks which

would effectively immobilize the joint whilst our sheep

were allowed to freely mobilize. Harrison et al.27 showed

the development of a predominantly indirect type enthesis

at 12 weeks which match the findings in the control group.

Most other tendon–bone healing models use a bone tunnel

which makes comparison with this study difficult.

We have used an ovine patellar tendon model to assesstendon–bone healing. Allen et al.28 showed that the ovinestifle joint is anatomically similar to the human knee, pos-sessing one distinct patellar tendon, with reduced medialand lateral retinacular expansions. In humans and sheep,the patellar tendon has no compensatory structures, unlikethose observed in other ruminants.28 As a result the entireextensor mechanism depends on attachment at the tendon–bone interface. Whilst anatomically equivalent, detachmentof the supraspinatus has a substantially lesser effect on gaitfunction as other components of the rotator cuff can com-pensate for its loss.29 Despite this model not precisely rep-resenting rotator cuff tears in humans, we feel that itenables more objective measures of functional recovery tobe assessed in a healing tendon–bone interface model.

Thirty three percent of animals in the control group

failed. These occurred within 6 weeks and were all due to

tendon avulsion from bone. In contrast, no failures were

observed in the DBM group. Despite this, no significant

difference was observed between the groups. We believe

this may be partly due to the small number of animals used

here, and postulate that further studies with increased group



size could yield statistical significance. The data suggests

the mode and timing of failure is similar to that reported

for rotator cuff surgery in humans5 and contributes to the

clinical relevance of this study.

Previous work has used force plate analysis as an indi-

rect measure of patellar tendon reattachment.21,30 The

forces encountered in the patellar tendon are related to the

peak vertical component of the GRFz in a quadrupedal

model.21 Early mobilization prevents weakness and stiff-

ness in a healing tendon–bone reconstruction.31 Our force

plate data show that the DBM group were able to mobilize

earlier than the control animals, and demonstrate superior

function at all time points. Early mobilization may be

enabled by DBM both mechanically and biologically. DBM

augmentation may dissipate the stresses encountered at the

interface over a wider area, thus reducing failure associated

with early weight bearing.

At 12 weeks, our mechanical testing showed no signifi-

cant difference between the UTS of the two groups, with

all samples failing in the tendon midsubstance. We postu-

late that at this time point, the UTS of the interface

exceeded that of tendon. These findings concur with those

of Rodeo et al. who showed that by 12 weeks the site of

failure moves from the attachment site to the tendon mid-

substance10 indicating that the true interface pullout

strength was greater than that recorded.

At 6 weeks, our control specimens displayed an imma-

ture, indirect-type enthesis, which by 12 weeks was inter-

spersed with regions of poorly organized fibrocartilage.

Studies using similar models to assess patellar tendon–bone

healing without biological augmentation have shown com-

parable healing patterns.24,26 Other models of tendon–bone

healing have looked primarily at ACL reconstructions in

dog and rabbit models, which exhibit differences in inter-

face morphology at different time points. These discrepan-

cies may be due to the differences in mechanical

environment, tendon–bone contact area, fixation method,

and species between the studies. We feel that our findings

are representative of a generic extra-articular tendon–bone

healing model.

In 1993 Rodeo et al.10 showed that increasing bone

formation at the healing tendon–bone enthesis leads to

increased pull-out strength of the resulting interface. Con-

sequently, subsequent studies have focused on improving

new bone growth at the tendon–bone interface. However,

the aim of tendon–bone healing is not to increase bone

growth, but to establish an enthesis with increasing min-

eralization through transitional fibrocartilaginous zones

between tendon and bone. The increase in fibrocartilage

and mineralized fibrocartilage observed in our DBM

group is of greater consequence to the developing inter-

face, and may lead to the recapitulation of a normal

enthesis. The absence of any differences in new bone for-

mation between the groups does not negate our functional

findings, which further substantiate this claim. During

endochondral ossification, the DBM scaffold may be

populated by mesenchymal cells which differentiate into

chondrocytes. The precise mechanism by which these

cells form layers of progressive mineralized tissue is

unknown, however BMP-2, 4,32,33 7,33 TGF-b1,34 and

IGF-134 have all been implicated in the signaling cascade.

The mechanical environment undoubtedly plays a crucial

role in tissue differentiation at the healing tendon–bone

interface, and in our model early healing and rejuvenation

of a structurally sound enthesis may allows faster func-

tional recovery which would help to promote tissue dif-

ferentiation.

In this study we have shown that DBM augmentation of

the healing patellar tendon–bone interface results in earlier

mobilization with fewer pull-out failures, and superior

functional and morphological recovery. A direct-type

enthesis and a FWB status of 95% was observed after ten-

don reattachment at 12 weeks. We believe that DBM aug-

mentation may improve functional and histological

recovery of tendon reattachments clinically.

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Tendon bone healing can be enhanced by demineralized bone matrix: A functional and histological study