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Enhanced bone formation in sheep vertebral bodies after minimally invasive treatment with a novel, PLGA fiber-reinforced brushite cement
Accepted Manuscript Title: Enhanced bone formation in sheep vertebral bodies after minimallyinvasive treatment with a novel, PLGA-fiber reinforced brushite cement Author: Stefan Maenz, Olaf Brinkmann, Elke Kunisch, Victoria Horbert, Francesca Gunnella, Sabine Bischoff, Harald Schubert, Andre Sachse, Long Xin, Jens Günster, Bernhard Illerhaus, Klaus D. Jandt, Jörg Bossert, Raimund W. Kinne, Matthias Bungartz PII: DOI: Reference:
S1529-9430(16)31066-X http://dx.doi.org/doi: 10.1016/j.spinee.2016.11.006 SPINEE 57204
To appear in:
The Spine Journal
Received date: Revised date: Accepted date:
30-5-2016 21-9-2016 9-11-2016
Please cite this article as: Stefan Maenz, Olaf Brinkmann, Elke Kunisch, Victoria Horbert, Francesca Gunnella, Sabine Bischoff, Harald Schubert, Andre Sachse, Long Xin, Jens Günster, Bernhard Illerhaus, Klaus D. Jandt, Jörg Bossert, Raimund W. Kinne, Matthias Bungartz, Enhanced bone formation in sheep vertebral bodies after minimally-invasive treatment with a novel, PLGA-fiber reinforced brushite cement, The Spine Journal (2016), http://dx.doi.org/doi: 10.1016/j.spinee.2016.11.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Enhanced bone formation in sheep vertebral bodies after minimally-invasive treatment with a
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novel, PLGA-fiber reinforced brushite cement
3
Stefan Maenza,#, Olaf Brinkmannbc,#, Elke Kunischc, Victoria Horbertc, Francesca Gunnellac, Sabine
4
Bischoffd, Harald Schubertd, Andre Sachseb, Long Xinc, Jens Günstere, Bernhard Illerhause, Klaus D.
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Jandta, Jörg Bosserta, Raimund W. Kinnec,* and Matthias Bungartzbc
6 7
a
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Jena, Löbdergraben 32, D-07743 Jena, Germany
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b
Chair of Materials Science, Otto Schott Institute of Materials Research, Friedrich Schiller University
Chair of Orthopedics, Department of Orthopedics, Jena University Hospital, Waldkrankenhaus
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“Rudolf Elle”, Klosterlausnitzer Str. 81, D-07607 Eisenberg, Germany
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c
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Waldkrankenhaus “Rudolf Elle”, Klosterlausnitzer Str. 81, D-07607 Eisenberg, Germany
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d
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D-07743 Jena, Germany
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e
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Germany.
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#
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*Corresponding author: Raimund W. Kinne; Experimental Rheumatology Unit, Department of
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Orthopedics, Jena University Hospital, Waldkrankenhaus “Rudolf Elle”, Klosterlausnitzer Str. 81, D-
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07607 Eisenberg; Germany. E-Mail: [email protected], phone: 0049 (0) 36691
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81228, fax: 0049 (0) 36691 81226.
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Acknowledgement
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We gratefully acknowledge the financial support by the Carl Zeiss Foundation (doctoral candidate
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scholarship to S.M.) and by the German Federal Ministry of Education and Research (BMBF FKZ
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0316205C to J.B and K.D.J.; BMBF FKZ 035577D, 0316205B, and 13N12601 to R.W.K).
Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital,
Institute of Laboratory Animal Sciences and Welfare, Jena University Hospital, Dornburger Str. 23,
Federal Institute for Materials Research and Testing (BAM), Unter den Eichen 87, D-12205 Berlin,
Both authors have equally contributed to the study.
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Funding: SM: Carl Zeiss Foundation (PhD scholarship); JB: German Federal Ministry of Education
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and Research (BMBF; FKZ 0316205C); KDJ: BMBF (FKZ 0316205C); RWK: BMBF (FKZ
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03577D, 0316205B, and 13N12601); LX: Department of Health of Zhejiang Province, Hangzhou,
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China (Research scholarship).
5 6
Abstract
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Background: Injectable, brushite-forming calcium phosphate cements (CPC) show potential for bone
8
replacement, but they exhibit low mechanical strength. This study tested a CPC reinforced with poly
9
(l-lactide-co-glycolide) acid (PLGA) fibers in a minimally-invasive, sheep lumbar vertebroplasty
10
model.
11
Purpose: To test the in vivo biocompatibility and osteogenic potential of a PLGA fiber-reinforced,
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brushite-forming CPC in a sheep large animal model.
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Study Design/Setting: Prospective experimental animal study
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Methods: Bone defects (diameter 5 mm) were placed in aged, osteopenic female sheep and left empty
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(L2) or injected with pure CPC (L3) or PLGA-fiber-reinforced CPC (L4; fiber diameter 25 µm; length
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1 mm; 10% (w/w)). Three and 9 months post-operation (n = 20 each), structural and functional CPC
17
effects on bone regeneration were documented ex vivo by osteodensitometry, histomorphometry,
18
micro-CT, and biomechanical testing.
19
Results: Addition of PLGA fibers enhanced CPC osteoconductivity and augmented bone formation.
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This was demonstrated by: i) significantly enhanced structural (bone volume/total volume; shown by
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micro-CT and histomorphometry; 3 and/or 9 months) and bone formation parameters (osteoid volume,
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osteoid surface; 9 months); ii) numerically enhanced bone mineral density (3 and 9 months) and
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biomechanical compression strength (9 months); and iii) numerically decreased bone erosion (eroded
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surface; 3 and 9 months).
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Conclusion: PLGA-fiber-reinforced CPC is highly biocompatible and its PLGA-fiber component
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enhanced bone formation. Also, PLGA fibers improve the mechanical properties of brittle CPC, with
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potential applicability in load-bearing areas.
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Keywords: Calcium phosphate cement, fiber reinforcement, bone regeneration, large animal model
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sheep, vertebroplasty, in vivo
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1. Introduction
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Kyphoplasty and vertebroplasty with injectable cements are commonly applied for minimally-invasive
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augmentation of osteoporotic vertebral compression fractures. Injectable poly(methyl methacrylate)
4
(PMMA) cements are frequently used, despite their lack of bioactivity and biodegradability.
5
Additional problems may be caused by their supra-physiological strength and Young’s modulus,
6
resulting in critical loads and subsequent fractures in adjacent vertebral bodies [1–4].
7
Calcium phosphate cements (CPC), first described in the 1980s [5, 6], may represent a promising
8
alternative to PMMA, since they are highly biocompatible, biodegradable, osteoconductive, and
9
characterized by a Young’s modulus comparable to that of cancellous bone [7–9]. Degradability and
10
mechanical properties of CPC are mostly governed by their composition. In general, apatite-forming
11
CPC show higher mechanical strength than brushite-forming CPC [7], whereas brushite-forming CPC
12
are of special interest due to their increased solubility and therefore degradability under physiological
13
conditions.
14
The mechanical strength and fracture toughness of CPC can be increased by modification with
15
reinforcing fibers. Different studies have described the use of non-resorbable fibers such as aramide
16
[10, 11], polyamide [12], polypropylene [13], glass [10], or carbon [10, 14], whereas others have
17
focused on the application of resorbable biodegradable fibers, e.g., polycaprolactone, polylactide,
18
polyglycolide, or different copolymers of the above [10, 15–29], as also reviewed elsewhere [30, 31].
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For a resorbable CPC, biodegradable fibers in conjunction with degradable calcium phosphate phases
20
are desirable. Several studies have addressed the use of biodegradable poly(l-lactide-co-glycolide) acid
21
(PLGA) suture materials with yarn diameters of 200 µm to 350 µm as biodegradable fiber
22
reinforcement for CPC [10, 16–25]. In general, the addition of PLGA fibers results in increased
23
strength and work of fracture (WOF) with increased fiber volume fraction [10, 16, 25] or fiber length
24
[10]. However, the large diameter of commercial PLGA suture materials limits their application in
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injectable CPC.
26
Own studies have recently described a PLGA fiber-reinforced, brushite-forming CPC, whose
27
injectability mainly depended on the length of the applied PLGA fibers (diameter 25 µm, [27, 29]). 4 Page 4 of 27
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The mechanical strength showed a maximum at a fiber content of 5% (w/w), while the WOF was
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further augmented by increasing the fiber content up to 7.5% (w/w). Tests on the biocompatibility in
3
vitro showed a transiently delayed cell growth on the fiber-reinforced CPC compared to the pure CPC
4
[27].
5
Several studies have reported the occurrence of inflammatory reactions or osteomyelitis after
6
placement of implants made of polylactide or especially polyglycolide [32–34]. The extent of the
7
inflammatory reaction appeared to increase with higher volumes of the implanted polymer. Therefore,
8
the biocompatibility of polylactide, polyglycolide or PLGA-containing CPC needs to be carefully
9
investigated.
10
To the best of our knowledge, PLGA-containing brushite-forming CPC have not been tested in vivo so
11
far, whereas some studies on the biocompatibility of PLGA-containing apatite-forming CPC [24, 34–
12
39] have shown that the bone formation was not negatively affected by the addition of PLGA. Link et
13
al. [34], on the other hand, have described a minimal inflammatory response to PLGA-containing
14
apatite-forming CPC.
15
Thus far, PLGA-containing CPC have been tested mostly in rats [34–36] and rabbits [37–39], but only
16
once in a large animal model [40]. Therefore, the aim of this study was to test the in vivo
17
biocompatibility and osteogenic poetential of PLGA fiber-reinforced, brushite-forming CPC in a sheep
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large animal model for lumbar ventrolateral vertebroplasty. For this purpose, PLGA fibers were
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incorporated into a commercially available, CE certified brushite-forming CPC.
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2. Materials and Methods
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2.1 Preparation of the fiber-reinforced CPC
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PLGA fibers with a diameter of 25 µm were prepared and characterized as recently described [27]. In
4
brief, PLGA fibers were extruded from the granulate material PURASORB PLG 1017 (Purac,
5
Gorinchem, Netherlands) using a mini extrusion system (RANDCASTLE EXTRUSION SYSTEMS
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INC, Cedar Grove, USA). Fibers were then cut to a length of 597 ± 362 µm using a cutting mill
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PULVERISETTE 19 (FRITSCH GmbH, Idar-Oberstein, Germany) with a 1 mm sieve insert.
8
CPC powder with a fiber content of 10% (w/w) was prepared by mixing the powder component of a
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CE certified brushite-forming CPC (JectOS+, Kasios, L’Union, France) with a defined amount of
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fibers with a magnetic stirrer for 30 minutes. Afterwards, the mixture was sieved to remove fiber
11
ravels. The liquid compound of JectOS+ was then added at a powder-to-liquid ratio of 2.2 g/ml.
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2.2 Surgical procedure
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Aged, osteopenic [41, 42] female sheep were used (40 Merino animals; 6 – 9 years old; 68 – 110 kg
14
body weight; for the osteopenic status see the comparative values for bone volume/total volume in
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young (age 2-4 years) and old sheep (6-9 years) in the supplementary data, Figure S1). Permission for
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the animal experiments was obtained from the governmental commission for animal protection, Free
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State of Thuringia, Germany (registration number: 02-036/11).
18
Surgery was performed as a ventrolateral vertebroplasty. In brief, the anesthetized animals (Isofluran,
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1.5-1.8 % (v/v), AbbVie, Ludwigshafen, Germany; Propofol, 0.2 mg/kg/h, Fresenius Kabi
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Deutschland GmbH, Bad Homburg, Germany) were optimally positioned on their left side under x-ray
21
control. The lower third of the fourth lumbar vertebral body (L4) was identified by x-ray (lateral
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plane) for correct longitudinal placement. After a skin incision of 10 × 10 mm, the drill (5 mm
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diameter; Stryker Leibinger GmbH, Freiburg, Germany) was advanced towards the center of the
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vertebral body under continuous x-ray control in two planes. The final depth of the drill channel was
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defined by the contralateral delimitation of the respective processus spinosus. Afterwards, the drill was
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removed and CPC with PLGA fibers was injected through a separate bone filler/pestle system
27
(Medtronic, Minneapolis, USA) under radiolucent control (Fig. 1 (a)). 6 Page 6 of 27
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The same procedure was repeated for the vertebral bodies L3 and L2 using separate skin incisions and
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access channels. Vertebral body L3 was filled with pure CPC, L2 was left without cement and served
3
as empty defect control. L1 was left untouched and served as normal control (Fig. 1 (a)).
4
After completion of surgery, animals were housed in separate pens for 1-2 weeks and then returned to
5
long-care paddocks for 3 months (short-term) or 9 months (long-term).
6
After 3 or 9 months the animals (n = 20 each) were sacrificed by i.v. injection of overdosed barbiturate
7
(Pentobarbital™, Essex Pharma GmbH, Munich, Germany) and subsequent application of magnesium
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sulfate (MgSO4, Dr. Paul Lohmann GmbH, Emmerthal, Germany). The lumbar spine was removed
9
using an oscillating bone saw, analyzed by a spiral computer tomography system Bright Speed
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Performix 1.6 SI with a final resolution of 1 mm (General Electric Healthcare, Munich, Germany), and
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kept frozen until further use.
12
The results for the effects of pure CPC have been partially reported in a publication focused on the
13
sheep vertebral defect model [43].
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2.3 Digital osteodensitometry
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Osteodensitometry was conducted using a software-guided, digital bone density measuring instrument
16
(DEXA QDR 4500 Elite™; Hologic, Waltham, MA, USA). To achieve an artifact-free
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osteodensitometry, lumbar spines were sawn into individual vertebrae (L1, L2, L3, and L4); the
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spinous and transverse processes, as well as the covering and base plates, were removed from all
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individual vertebrae (final height in each case 15 mm). Osteodensitometric measurements were
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therefore limited to the central, spongious area of the vertebral bodies. A rectangular region of interest
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with a defined size of 9 x 11 mm served as measuring field (Fig. 1 (a)). The high density areas of the
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injected cement were excluded from quantification.
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2.4 Micro computed tomography (micro-CT)
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For the acquisition of 3-D images, an X-RAY WorX 225kV tube micro-CT system (X-RAY WorX
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GmbH, Garbsen, Germany) with a flat panel detector was used (PerkinElmer 1621; CsJ as scintillator;
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2048 × 2048 pixel; PerkinElmer, Waltham, USA). The following settings were used: 100 kV, 380 μA, 7 Page 7 of 27
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and a pre-filter of 0.5 mm copper. The shadow images had a size of 1951 × 1813 pixels, and 3600
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images were recorded for a full 360° turn (duration 6 s; average of 3 × 2 s). A total of 8 frozen samples
3
were analyzed under dry ice within 6 h, resulting in a voxel size of 66.6 μm after reconstruction with
4
the original Feldkamp code (Fig. 2).
5
Quantitative analysis of the micro-CT data was carried out using the 3-D software VGSTUDIO MAX
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2.2 (Volume Graphics GmbH, Heidelberg, Germany) and applying half-cylinders with a radius of 2.5,
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3, 3.5, 4, 4.5, and 5 mm (Fig. 3 (a) and (b)). The longitudinal axis and length of the half cylinders for
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each individual vertebral body were defined on the basis of the respective parameters of the drill
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channel. The exact 3-D position of the half cylinders (e.g., rotation and depth) was chosen to exclude
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cortical bone. Bone volume (BV) and total volume (TV) were separately determined by global
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threshold determination in the drill channel (maximal radius of 2.5 mm) and adjacent half cylinder
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segments (principle of onion shell). For this purpose, the respective maxima of grey values for soft
13
tissue, bone tissue, and cement were determined and the means between the maxima were used as a
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threshold for the volume determination of the individual components. The bone volume/total volume
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(BV/TV) was subsequently calculated.
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2.5 Histology and histomorphometry
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After cutting the lumbar vertebral bodies into two parts directly along the axis of the cement injection
18
channel, the analyses were carried out using two different types of histological sections: i) decalcified
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paraffin sections stained by hematoxylin-eosin [44]; or ii) plastic-embedded sections obtained by
20
fixation in acetone and dehydration in ascending alcohol series without demineralization. Static
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histomorphometry of the lumbar vertebral bodies (L1-L4) was performed according to published
22
procedures [45–47]. For this purpose, the samples were embedded in Technovit 9100 according to the
23
instructions of the supplier (Heraeus Kulzer, Wehrheim, Germany; [48]). Sections were then cut and
24
ground to a thickness of approx. 7 μm. For static histomorphometry, the sections were stained with
25
trichrome stain according to Masson-Goldner [44].
26
Static histomorphometry of each individual vertebral body in the immediate vicinity of the injection
27
channel was performed using a standard microscope (Axiovert 200 M, Carl Zeiss Microimaging 8 Page 8 of 27
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GmbH, Oberkochen, Germany) with a 200-fold magnification, and a Merz counting reticule [47, 49].
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The following 7 parameters were calculated according to the recommendations of the International
3
Committee of the American Society for Bone and Mineral Research (ASBMR) [45, 46]: Bone
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volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), osteoid
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volume (OV/BV), osteoid surface (OS/BS), osteoid thickness (O.Th), and eroded Surface (ES/BS).
6
Detailed information on the calculation of the parameters can be found in the supplementary data
7
(including Fig. S2).
8
2.6 Biomechanical testing
9
Biomechanical compressive strength measurements were conducted using a universal material testing
10
machine (FPG 7/20-010™; Kögel, Leipzig, Germany) and the corresponding software (FRK Quicktest
11
2004.01™). For biomechanical testing, frozen cancellous bone cylinders (10 mm diameter x 15 mm
12
height) were obtained from the central part of the vertebral bodies (L1-L4) using a surgical diamond
13
hollow milling cutter (diameter 10 mm). After a defined thawing time of exactly 30 minutes, samples
14
were semi-confined in 2 semilunar clamps (minimal inner diameter of 10.1 mm; length 9.8 mm) and
15
then compressed along their longitudinal axis until fracturing. This axis was chosen because it
16
represents the main loading axis in humans and is therefore of major interest for future clinical
17
application.
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2.7 Statistics
19
The data were expressed as means ± standard errors of mean. The Wilcoxon test was used to analyze
20
the results of paired samples (samples from the same animals) for statistically significant differences.
21
Differences between 3 and 9 month animals were analyzed for statistical significance using the Mann-
22
Whitney U test. Non-parametric statistical tests were used in the present study due to a lack of normal
23
distribution of the data in a high percentage of the experimental groups. For all tests, the level of
24
significance was set at p ≤ 0.05. All statistical tests were performed using the Sigmaplot software
25
release 13.0 (Systat Software Inc., Chicago, USA).
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On the basis of a previous study [43] comparing the effects of an injection of pure CPC to an empty
27
defect, an effect size of 0.7 was calculated, i.e. considerably higher than a medium effect size of 0.5. 9 Page 9 of 27
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Using this effect size in a two-tailed Wilcoxon signed-rank test (matched pairs) with an α of 0.05 and a
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power of 0.80, a sample number of 19 was computed (G*Power 3.1.9.2). In order to address a
3
potentially lower difference between pure and fiber-reinforced CPC in the present study, the sample
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number was raised to n = 20 for each of the 2 time points 3 months and 9 months.
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3. Results
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3.1 Osteodensitometry
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At 3 and 9 months, the bone mineral density (BMD) in vertebral bodies with an empty defect (L2), as
4
well as in both CPC-treated vertebral bodies (L3 and L4; ± PLGA), was significantly higher compared
5
to the untreated control (L1; Fig. 1b). Statistically significant differences between vertebral bodies
6
treated with either pure CPC (L3) or CPC with PLGA fibers (L4) were not observed (Fig. 1b).
7
Interestingly, at 3 months the vertebral bodies treated with CPC with PLGA fibers (L4) also showed a
8
significant BMD increase compared to the empty defect (L2). In addition, the BMD numerically
9
decreased from 3 to 9 months for all three treated vertebral bodies (L2, L3 and L4), but without
10 11
significant differences between the 3 and 9 months values. 3.2 Micro computed tomography
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In representative 9-month micro-CT images of untreated controls (L1) and treated vertebral bodies
13
(L2, L3, and L4), empty defects (L2) showed incomplete bone healing and remodeling mainly
14
restricted to the original drill channel (Fig.2 (b)). In contrast, both CPC-treated vertebral bodies (L3
15
and L4; ± PLGA) showed an increased spongiosa density directly adjacent to the edge of the drill
16
channel, but still incomplete bone healing and remodeling (Fig. 2 (c) and (d)).
17
At 3 months, quantitative evaluation of the micro-CT data revealed a significantly increased bone
18
volume/total volume as compared to untreated controls (L1) in vertebral bodies with an empty defect
19
(L2) up to a distance of 1 mm from the original defect (Fig. 3 (c)). For higher distances, this bone
20
volume/total volume matched that of the untreated controls (L1). In contrast, the implantation of pure
21
CPC (L3) or CPC with PLGA fibers (L4) significantly enhanced bone formation up to a distance of at
22
least 2.5 mm. At the high distances of 2 and 2.5 mm, this osteoconductive effect was significantly
23
higher for CPC with PLGA fibers (L4) than for pure CPC (L3; Fig. 3 (c)).
24
At 9 months, there was still a significantly increased bone volume/total volume as compared to
25
untreated controls (L1) in vertebral bodies with an empty defect (L2) up to a distance of 0.5 mm from
26
the original defect (Fig. 3 (d)). Also, the implantation of pure CPC (L3) or CPC with PLGA fibers
27
(L4) still significantly enhanced bone formation around the drill channel, however only up to a 11 Page 11 of 27
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distance of 1 mm and without statistically significant differences between pure CPC (L3) and CPC
2
with PLGA fibers (L4). The values of bone volume/total volume were generally lower after 9 months
3
compared to 3 months, with significant differences for pure CPC (L3) in the immediate vicinity of the
4
drill channel and for the CPC with PLGA fibers (L4) starting at a distance of 1 mm from the original
5
defect (Fig. 3 (d)).
6
3.3 Histomorphometry
7
In representative 9 month histology images of untreated controls (L1) and treated vertebral bodies (L2,
8
L3 and L4), empty defects (L2) confirmed incomplete healing and remodeling mainly in the area of
9
the original defect (Fig. 4 (d)-(f)) as observed in the micro-CT sections. In contrast, the dense bony
10
layer in the vicinity of the CPC implants (L3 and L4; ± PLGA) was also visible in the histology
11
images (Fig. 4 (g)-(m)). Also, there was a clear formation of bone bridges and islands in the cement
12
region of vertebral bodies treated with either pure CPC or CPC with PLGA fibers (L3 and L4; Fig. 4
13
(h), (i), (l) and (m)). There were no signs of inflammatory infiltration close to or distant from the
14
injection channel in either the CPC (L3) or the CPC with PLGA fiber (L4) group at any time point.
15
At 3 and 9 months, bone volume/total volume and trabecular thickness in all treated vertebral bodies
16
(L2, L3, and L4) were significantly higher than in untreated controls (L1; Fig. 5 (a) and (b)). Also,
17
CPC-treated vertebral bodies (L3 and L4; ± PLGA) mostly showed significantly higher values than
18
empty defects (L2; Fig. 5 (a) and (b)). Notably, after 9 months the bone volume/total volume for CPC
19
with PLGA fibers (L4) was significantly higher than that for pure CPC (L3; Fig. 5 (a)), whereas the
20
trabecular thickness at 3 and 9 months was significantly lower for the CPC with PLGA fibers (L4; Fig.
21
5 (b)).
22
In contrast to the findings for bone volume/total volume and trabecular thickness, the trabecular
23
number for all treated vertebral bodies (L2, L3 and L4) was significantly lower than that for untreated
24
controls (L1); in addition, the trabecular number for pure CPC (L3) was significantly lower than that
25
for empty defects (L2; Fig. 5 (c)). In contrast, the trabecular number for the CPC with PLGA fibers
26
was significantly higher than that for empty defects at 9 months (L2) and significantly higher than that
27
for pure CPC at 3 and 9 months (L3; Fig. 5 (c)). 12 Page 12 of 27
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At 9 months, the bone formation parameters osteoid volume, osteoid surface and osteoid thickness for
2
the CPC with PLGA fibers (L4) were always numerically higher than those for all other vertebral
3
bodies (L1-L3), with statistically significant differences compared to either untreated vertebral bodies
4
(L1; osteoid surface, osteoid thickness) or vertebral bodies treated with pure CPC (L3; osteoid volume,
5
osteoid surface; Fig. 6 (a)-(c)). In parallel, the bone degradation parameter eroded surface was
6
significantly lower for CPC with PLGA fibers (L4) than that for untreated controls (L1; Fig. 6 (d)).
7
3.4 Biomechanical testing
8
At 3 and 9 months, the biomechanical compression strength for all treated vertebral bodies (L2, L3,
9
and L4) was numerically higher than that for untreated controls (L1; Fig. 7). Notably, at 9 months
10
CPC with PLGA fibers (L4) showed significantly higher values than untreated controls (L1) and
11
empty defects (L2; Fig. 7).
13 Page 13 of 27
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4. Discussion
2
The main aim of the present study was to investigate the biocompatibility of the PLGA fiber
3
component of the fiber-reinforced, brushite-forming CPC. The results were consistent in indicating the
4
following aspects: i) the PLGA fibers had no negative effect on bone formation at any time point or
5
with any of the analyses; ii) the presence of PLGA fibers even enhanced the osteoconductivity of the
6
CPC and led to significantly or numerically increased parameters of bone formation. The latter was
7
demonstrated by significantly enhanced structural parameters (bone volume/total volume at 3 and/or 9
8
months) and bone formation parameters (osteoid volume and osteoid surface at 9 months). The BMD
9
was also enhanced at 3 and 9 months, albeit not at a statistically significant level; the biomechanical
10
compression strength was enhanced at 9 months; and, finally, bone erosion as illustrated by the degree
11
of eroded surface was also numerically reduced at 3 and 9 months. Thus the novel PLGA fiber-
12
reinforced, brushite-forming CPC is highly biocompatible in a sheep lumbar vertebroplasty model, and
13
its PLGA fiber component appears to even support bone formation. These characteristics are very
14
promising in view of using PLGA fibers for the improvement of mechanical properties of inherently
15
brittle CPC and thus for potential application in the therapy of vertebral fractures in load-bearing areas
16
[27, 29, 50].
17
Concerning the main time period of osteoconductivity of the PLGA fibers, some of the parameters
18
indicated predominantly early effects (i.e., BMD, bone volume/total volume in micro-CT and
19
histomorphometry), whereas all bone formation parameters and the biomechanical properties pointed
20
to later effects. In addition, the trabecular structure of the newly formed bone inside or adjacent to the
21
injection channel at 9 months still appeared rather immature with few, but relatively thick trabeculae
22
(Fig. 4). This may have been even more pronounced after 3 months, as indicated by a significantly
23
higher bone volume/total volume in both micro-CT and histomorphometry, as well as by a higher
24
trabecular thickness but lower trabecular number when comparing the 3 months groups to the 9
25
months groups. These findings are in good agreement with previous reports on the presence of only
26
partially remodeled new bone 12 weeks after implantation of a hydroxyapatite-forming CPC
27
containing 31% (w/w) PLGA microspheres into the mandible of adult minipigs [40], as well as with
28
common knowledge on the temporal dynamics of bone healing and remodeling [51]. The present 14 Page 14 of 27
1
findings indicate on one hand that long-term bone remodeling in the vicinity of the injected CPC with
2
fibers (as in the case of the pure CPC) is still ongoing at 9 months. On the other hand, they show that
3
the osteoconductive effects of CPC and/or PLGA fibers may be transient.
4
The differential effects of the PLGA fibers on the osteoconductivity of the composite CPC likely
5
depend on their resorbability in vivo. Depending on their composition, physical shape/size, %
6
proportion, and connectivity (among other factors), PLGA fibers are rapidly resorbed in vivo, resulting
7
in resorption of PLGA-containing CPC between 10% and 90% at 12 weeks [24, 35, 37, 39]. Some
8
evidence of partial degradation of the CPC with fibers was also observed in the present study, with
9
significantly lower values in comparison to pure CPC at distances of 1.0 and 1.5 mm from the drill
10
channel after 3 months, and directly in the cement cylinder after 9 months (data not shown). This
11
indicates that gradual resorption of the PLGA from the periphery to the drill channel may contribute to
12
the enhanced osteoconductivity of the CPC with fibers.
13
Interestingly, significant osteoconductive effects of both CPC and/or CPC with fibers were observed
14
up to a distance of 2.5 mm from the edge of the injection channel, whereas such ‘remote’ effects were
15
limited to a distance of 1 mm in the case of the empty defects. This is compatible with a high
16
degradability of the brushite-forming CPC used in the present study, as well as with the suggested
17
influence of free calcium and inorganic phosphates on the function of osteoprogenitor cells and
18
osteoblasts, and thus on bone formation [8, 52].
19
In the present study, there were no signs of inflammatory infiltration after the use of CPC or CPC
20
enhanced with PLGA fibers at any time point. This is in clear contrast to some studies showing
21
moderate to strong inflammatory reactions following implantation of PGA or PLGA material into
22
bone [32–34]. In these studies, the extent of the inflammatory reaction appeared to increase with a
23
higher volume or proportion of implanted polymer, either PGA or PLGA. Several different factors are
24
believed to contribute to in situ side effects of degradable polymers, including size, location, time
25
point after implantation, and composition [53, 54]. On the other hand, several studies, including our
26
own, found no evidence of inflammation 12 weeks following bone implantation of CPC/PGA or
27
CPC/PLGA composites with considerable percentages of polymers, showing that the composites can 15 Page 15 of 27
1
be carefully designed to prevent unfavorable tissue reactions [36, 55]. The presence of inflammatory
2
cells at early time points, e.g., 4 weeks, cannot be totally excluded [36, 40], but in the present study –
3
even if present – they did not seem to result in negative long-term effects.
4
In general, vertebroplasty/kyphoplasty of vertebral bodies using PMMA or CPC bears the risk of
5
intravascular leakage potentially leading to pulmonary cement embolism and/or arterial blood pressure
6
alterations [56]. However, in a previous own study there were no in vivo signs of systemic
7
intravascular leakage after the injection of CPC [57]. In addition, local intravascular leakage after in
8
vivo application of CPC was only observed in a very limited percentage of vertebral bodies (approx.
9
10%) versus 53% in the case of a high-viscosity PMMA cement (Kyphon® HV-R®12, Medtronic
10
Inc., Milan, Italy). Finally, both pure and fiber-reinforced CPC ex vivo showed a significantly lower
11
intravertebral extrusion into the bone marrow than the high-viscosity PMMA cement [57]. These
12
findings rather argue for a decreased risk of the new CPC material for intravascular leakage.
13
Limitations of the present study
14
In the current study, pure CPC was injected into L3 and CPC with PLGA fibers was injected into L4.
15
Therefore, systematic inter-group statistical significant difference based on the experimental design
16
cannot be totally excluded. However, there were no statistically significant differences in structural
17
bone parameters between L3 and L4 of young sheep (2-4 years; see supplementary data, Figure S3). It
18
is thus not very likely (although not impossible) that systematic differences between the bone
19
regeneration ability in L3 and L4 have biased the results of the present study.
20
Structural and functional effects of CPC and with PLGA fibers on bone regeneration were tested in
21
aged sheep (6-9 years). These animals showed significantly reduced structural histomorphometric
22
parameter compared to young sheep (see supplementary data, Figure S1 and [42]) and were therefore
23
referred to as osteopenic. However, it is not clear whether the significant enhancement of bone
24
formation by the PLGA-fiber component observed in the present study is completely transferable to
25
human osteoporosis. A first step to answer this question may be the use of an ovine model with
26
induced osteoporosis, which, however, requires long-term and costly preparation of the animals [58].
27 16 Page 16 of 27
1
5. Conclusion
2
The fiber component of a PLGA fiber-reinforced, brushite-forming CPC not only lacked negative or
3
toxic effects, but it significantly enhanced bone formation at the vertebral site of injection. The
4
positive effects of the PLGA fibers on bone formation and the augmentation of the mechanical
5
properties of the resulting CPC [27, 29] may thus allow the development of optimized composite CPC
6
for potential application in vertebroplasty or kyphoplasty in (load-bearing) bone defects. One
7
particular advantage of the present composite CPC for future clinical development may be the usage of
8
2 components already CE certified as bone cement and suture material. Finally, the large animal
9
vertebroplasty model in sheep appeared well-suitable for the testing of vertebral augmentation with
10
resorbable and osteoconductive CPC.
17 Page 17 of 27
1
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Figure captions
2
Figure 1: (a) Control of CPC placement by conventional computer tomography; definition of regions
3
of interests (ROI) for osteodensitometry; (b) bone mineral density (BMD) 3 months and 9 months
4
after surgery (n = 20 each),as determined by osteodensitometry in differently treated lumbar vertebral
5
bodies. * p ≤ 0.05 vs. control (L1); + p ≤ 0.05 vs. empty defect (L2).
6
Figure 2: Representative images from micro computed tomography (micro-CT) of differently treated
7
lumbar vertebral bodies 9 months after surgery: (a) untreated control (L1); (b) empty defect (L2); (c)
8
CPC (L3); (d) CPC with PLGA.
9
Figure 3: Quantitative evaluation of the micro computed tomography (micro-CT) of differently treated
10
lumbar vertebral bodies: (a) and (b) schematic representation of the half-cylinders with a radius of 2.5,
11
3, 3.5, 4, 4.5, and 5 mm used for quantitative evaluation; (c) bone volume/total volume (BV/TV) after
12
3 months; (d) bone volume/total volume (BV/TV) after 9 months. * p ≤ 0.05 vs. control (L1); + p ≤
13
0.05 vs. empty defect (L2); § p ≤ 0.05 vs. CPC (L3); # p ≤ 0.05 vs. 3 months.
14
Figure 4: Representative histology images of differently treated, paraffin-embedded lumbar vertebral
15
bodies 9 months after surgery stained by hematoxylin-eosin: (a-c) untreated control (L1); (d-f) empty
16
defect (L2); (e-i) CPC (L3); (k-m) CPC with PLGA. Inserts in (a, b, d, e, g, h, k, and l) indicate the
17
regions shown in a higher magnifications in (b, c, e, f, h, i, l and m), respectively.
18
Figure 5: Structural bone parameters 3 months and 9 months after surgery (n = 20 each), as determined
19
by static histomorphometry in differently treated lumbar vertebral bodies: (a) bone volume/total
20
volume (BV/TV); (b) trabecular thickness (Tb.Th); (c) trabecular number (Tb.N). * p ≤ 0.05 vs.
21
control (L1); + p ≤ 0.05 vs. empty defect (L2); § p ≤ 0.05 vs. CPC (L3); # p ≤ 0.05 vs. 3 months.
22
Figure 6: Bone formation and erosion parameters 3 months and 9 months after surgery (n = 20 each),
23
as determined by static histomorphometry in differently treated lumbar vertebral bodies: (a) osteoid
24
volume (OV/BV); (b) osteoid surface (OS/BS); (c) osteoid thickness (O.Th); (d) eroded surface
25
(ES/BS). * p ≤ 0.05 vs. control (L1); § p ≤ 0.05 vs. CPC (L3); # p ≤ 0.05 vs. 3 months.
25 Page 25 of 27
1
Figure 7: Biomechanical compressive strength of cancellous bone 3 months and 9 months after
2
surgery (n = 20 each) in differently treated lumbar vertebral bodies. * p ≤ 0.05 vs. control (L1); + p ≤
3
0.05 vs. empty (L2).
4
26 Page 26 of 27
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27 Page 27 of 27
S1529-9430(16)31066-X http://dx.doi.org/doi: 10.1016/j.spinee.2016.11.006 SPINEE 57204
To appear in:
The Spine Journal
Received date: Revised date: Accepted date:
30-5-2016 21-9-2016 9-11-2016
Please cite this article as: Stefan Maenz, Olaf Brinkmann, Elke Kunisch, Victoria Horbert, Francesca Gunnella, Sabine Bischoff, Harald Schubert, Andre Sachse, Long Xin, Jens Günster, Bernhard Illerhaus, Klaus D. Jandt, Jörg Bossert, Raimund W. Kinne, Matthias Bungartz, Enhanced bone formation in sheep vertebral bodies after minimally-invasive treatment with a novel, PLGA-fiber reinforced brushite cement, The Spine Journal (2016), http://dx.doi.org/doi: 10.1016/j.spinee.2016.11.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Enhanced bone formation in sheep vertebral bodies after minimally-invasive treatment with a
2
novel, PLGA-fiber reinforced brushite cement
3
Stefan Maenza,#, Olaf Brinkmannbc,#, Elke Kunischc, Victoria Horbertc, Francesca Gunnellac, Sabine
4
Bischoffd, Harald Schubertd, Andre Sachseb, Long Xinc, Jens Günstere, Bernhard Illerhause, Klaus D.
5
Jandta, Jörg Bosserta, Raimund W. Kinnec,* and Matthias Bungartzbc
6 7
a
8
Jena, Löbdergraben 32, D-07743 Jena, Germany
9
b
Chair of Materials Science, Otto Schott Institute of Materials Research, Friedrich Schiller University
Chair of Orthopedics, Department of Orthopedics, Jena University Hospital, Waldkrankenhaus
10
“Rudolf Elle”, Klosterlausnitzer Str. 81, D-07607 Eisenberg, Germany
11
c
12
Waldkrankenhaus “Rudolf Elle”, Klosterlausnitzer Str. 81, D-07607 Eisenberg, Germany
13
d
14
D-07743 Jena, Germany
15
e
16
Germany.
17
#
18
*Corresponding author: Raimund W. Kinne; Experimental Rheumatology Unit, Department of
19
Orthopedics, Jena University Hospital, Waldkrankenhaus “Rudolf Elle”, Klosterlausnitzer Str. 81, D-
20
07607 Eisenberg; Germany. E-Mail: [email protected], phone: 0049 (0) 36691
21
81228, fax: 0049 (0) 36691 81226.
22
Acknowledgement
23
We gratefully acknowledge the financial support by the Carl Zeiss Foundation (doctoral candidate
24
scholarship to S.M.) and by the German Federal Ministry of Education and Research (BMBF FKZ
25
0316205C to J.B and K.D.J.; BMBF FKZ 035577D, 0316205B, and 13N12601 to R.W.K).
Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital,
Institute of Laboratory Animal Sciences and Welfare, Jena University Hospital, Dornburger Str. 23,
Federal Institute for Materials Research and Testing (BAM), Unter den Eichen 87, D-12205 Berlin,
Both authors have equally contributed to the study.
1 Page 1 of 27
1
Funding: SM: Carl Zeiss Foundation (PhD scholarship); JB: German Federal Ministry of Education
2
and Research (BMBF; FKZ 0316205C); KDJ: BMBF (FKZ 0316205C); RWK: BMBF (FKZ
3
03577D, 0316205B, and 13N12601); LX: Department of Health of Zhejiang Province, Hangzhou,
4
China (Research scholarship).
5 6
Abstract
7
Background: Injectable, brushite-forming calcium phosphate cements (CPC) show potential for bone
8
replacement, but they exhibit low mechanical strength. This study tested a CPC reinforced with poly
9
(l-lactide-co-glycolide) acid (PLGA) fibers in a minimally-invasive, sheep lumbar vertebroplasty
10
model.
11
Purpose: To test the in vivo biocompatibility and osteogenic potential of a PLGA fiber-reinforced,
12
brushite-forming CPC in a sheep large animal model.
13
Study Design/Setting: Prospective experimental animal study
14
Methods: Bone defects (diameter 5 mm) were placed in aged, osteopenic female sheep and left empty
15
(L2) or injected with pure CPC (L3) or PLGA-fiber-reinforced CPC (L4; fiber diameter 25 µm; length
16
1 mm; 10% (w/w)). Three and 9 months post-operation (n = 20 each), structural and functional CPC
17
effects on bone regeneration were documented ex vivo by osteodensitometry, histomorphometry,
18
micro-CT, and biomechanical testing.
19
Results: Addition of PLGA fibers enhanced CPC osteoconductivity and augmented bone formation.
20
This was demonstrated by: i) significantly enhanced structural (bone volume/total volume; shown by
21
micro-CT and histomorphometry; 3 and/or 9 months) and bone formation parameters (osteoid volume,
22
osteoid surface; 9 months); ii) numerically enhanced bone mineral density (3 and 9 months) and
23
biomechanical compression strength (9 months); and iii) numerically decreased bone erosion (eroded
24
surface; 3 and 9 months).
2 Page 2 of 27
1
Conclusion: PLGA-fiber-reinforced CPC is highly biocompatible and its PLGA-fiber component
2
enhanced bone formation. Also, PLGA fibers improve the mechanical properties of brittle CPC, with
3
potential applicability in load-bearing areas.
4
Keywords: Calcium phosphate cement, fiber reinforcement, bone regeneration, large animal model
5
sheep, vertebroplasty, in vivo
3 Page 3 of 27
1
1. Introduction
2
Kyphoplasty and vertebroplasty with injectable cements are commonly applied for minimally-invasive
3
augmentation of osteoporotic vertebral compression fractures. Injectable poly(methyl methacrylate)
4
(PMMA) cements are frequently used, despite their lack of bioactivity and biodegradability.
5
Additional problems may be caused by their supra-physiological strength and Young’s modulus,
6
resulting in critical loads and subsequent fractures in adjacent vertebral bodies [1–4].
7
Calcium phosphate cements (CPC), first described in the 1980s [5, 6], may represent a promising
8
alternative to PMMA, since they are highly biocompatible, biodegradable, osteoconductive, and
9
characterized by a Young’s modulus comparable to that of cancellous bone [7–9]. Degradability and
10
mechanical properties of CPC are mostly governed by their composition. In general, apatite-forming
11
CPC show higher mechanical strength than brushite-forming CPC [7], whereas brushite-forming CPC
12
are of special interest due to their increased solubility and therefore degradability under physiological
13
conditions.
14
The mechanical strength and fracture toughness of CPC can be increased by modification with
15
reinforcing fibers. Different studies have described the use of non-resorbable fibers such as aramide
16
[10, 11], polyamide [12], polypropylene [13], glass [10], or carbon [10, 14], whereas others have
17
focused on the application of resorbable biodegradable fibers, e.g., polycaprolactone, polylactide,
18
polyglycolide, or different copolymers of the above [10, 15–29], as also reviewed elsewhere [30, 31].
19
For a resorbable CPC, biodegradable fibers in conjunction with degradable calcium phosphate phases
20
are desirable. Several studies have addressed the use of biodegradable poly(l-lactide-co-glycolide) acid
21
(PLGA) suture materials with yarn diameters of 200 µm to 350 µm as biodegradable fiber
22
reinforcement for CPC [10, 16–25]. In general, the addition of PLGA fibers results in increased
23
strength and work of fracture (WOF) with increased fiber volume fraction [10, 16, 25] or fiber length
24
[10]. However, the large diameter of commercial PLGA suture materials limits their application in
25
injectable CPC.
26
Own studies have recently described a PLGA fiber-reinforced, brushite-forming CPC, whose
27
injectability mainly depended on the length of the applied PLGA fibers (diameter 25 µm, [27, 29]). 4 Page 4 of 27
1
The mechanical strength showed a maximum at a fiber content of 5% (w/w), while the WOF was
2
further augmented by increasing the fiber content up to 7.5% (w/w). Tests on the biocompatibility in
3
vitro showed a transiently delayed cell growth on the fiber-reinforced CPC compared to the pure CPC
4
[27].
5
Several studies have reported the occurrence of inflammatory reactions or osteomyelitis after
6
placement of implants made of polylactide or especially polyglycolide [32–34]. The extent of the
7
inflammatory reaction appeared to increase with higher volumes of the implanted polymer. Therefore,
8
the biocompatibility of polylactide, polyglycolide or PLGA-containing CPC needs to be carefully
9
investigated.
10
To the best of our knowledge, PLGA-containing brushite-forming CPC have not been tested in vivo so
11
far, whereas some studies on the biocompatibility of PLGA-containing apatite-forming CPC [24, 34–
12
39] have shown that the bone formation was not negatively affected by the addition of PLGA. Link et
13
al. [34], on the other hand, have described a minimal inflammatory response to PLGA-containing
14
apatite-forming CPC.
15
Thus far, PLGA-containing CPC have been tested mostly in rats [34–36] and rabbits [37–39], but only
16
once in a large animal model [40]. Therefore, the aim of this study was to test the in vivo
17
biocompatibility and osteogenic poetential of PLGA fiber-reinforced, brushite-forming CPC in a sheep
18
large animal model for lumbar ventrolateral vertebroplasty. For this purpose, PLGA fibers were
19
incorporated into a commercially available, CE certified brushite-forming CPC.
20
5 Page 5 of 27
1
2. Materials and Methods
2
2.1 Preparation of the fiber-reinforced CPC
3
PLGA fibers with a diameter of 25 µm were prepared and characterized as recently described [27]. In
4
brief, PLGA fibers were extruded from the granulate material PURASORB PLG 1017 (Purac,
5
Gorinchem, Netherlands) using a mini extrusion system (RANDCASTLE EXTRUSION SYSTEMS
6
INC, Cedar Grove, USA). Fibers were then cut to a length of 597 ± 362 µm using a cutting mill
7
PULVERISETTE 19 (FRITSCH GmbH, Idar-Oberstein, Germany) with a 1 mm sieve insert.
8
CPC powder with a fiber content of 10% (w/w) was prepared by mixing the powder component of a
9
CE certified brushite-forming CPC (JectOS+, Kasios, L’Union, France) with a defined amount of
10
fibers with a magnetic stirrer for 30 minutes. Afterwards, the mixture was sieved to remove fiber
11
ravels. The liquid compound of JectOS+ was then added at a powder-to-liquid ratio of 2.2 g/ml.
12
2.2 Surgical procedure
13
Aged, osteopenic [41, 42] female sheep were used (40 Merino animals; 6 – 9 years old; 68 – 110 kg
14
body weight; for the osteopenic status see the comparative values for bone volume/total volume in
15
young (age 2-4 years) and old sheep (6-9 years) in the supplementary data, Figure S1). Permission for
16
the animal experiments was obtained from the governmental commission for animal protection, Free
17
State of Thuringia, Germany (registration number: 02-036/11).
18
Surgery was performed as a ventrolateral vertebroplasty. In brief, the anesthetized animals (Isofluran,
19
1.5-1.8 % (v/v), AbbVie, Ludwigshafen, Germany; Propofol, 0.2 mg/kg/h, Fresenius Kabi
20
Deutschland GmbH, Bad Homburg, Germany) were optimally positioned on their left side under x-ray
21
control. The lower third of the fourth lumbar vertebral body (L4) was identified by x-ray (lateral
22
plane) for correct longitudinal placement. After a skin incision of 10 × 10 mm, the drill (5 mm
23
diameter; Stryker Leibinger GmbH, Freiburg, Germany) was advanced towards the center of the
24
vertebral body under continuous x-ray control in two planes. The final depth of the drill channel was
25
defined by the contralateral delimitation of the respective processus spinosus. Afterwards, the drill was
26
removed and CPC with PLGA fibers was injected through a separate bone filler/pestle system
27
(Medtronic, Minneapolis, USA) under radiolucent control (Fig. 1 (a)). 6 Page 6 of 27
1
The same procedure was repeated for the vertebral bodies L3 and L2 using separate skin incisions and
2
access channels. Vertebral body L3 was filled with pure CPC, L2 was left without cement and served
3
as empty defect control. L1 was left untouched and served as normal control (Fig. 1 (a)).
4
After completion of surgery, animals were housed in separate pens for 1-2 weeks and then returned to
5
long-care paddocks for 3 months (short-term) or 9 months (long-term).
6
After 3 or 9 months the animals (n = 20 each) were sacrificed by i.v. injection of overdosed barbiturate
7
(Pentobarbital™, Essex Pharma GmbH, Munich, Germany) and subsequent application of magnesium
8
sulfate (MgSO4, Dr. Paul Lohmann GmbH, Emmerthal, Germany). The lumbar spine was removed
9
using an oscillating bone saw, analyzed by a spiral computer tomography system Bright Speed
10
Performix 1.6 SI with a final resolution of 1 mm (General Electric Healthcare, Munich, Germany), and
11
kept frozen until further use.
12
The results for the effects of pure CPC have been partially reported in a publication focused on the
13
sheep vertebral defect model [43].
14
2.3 Digital osteodensitometry
15
Osteodensitometry was conducted using a software-guided, digital bone density measuring instrument
16
(DEXA QDR 4500 Elite™; Hologic, Waltham, MA, USA). To achieve an artifact-free
17
osteodensitometry, lumbar spines were sawn into individual vertebrae (L1, L2, L3, and L4); the
18
spinous and transverse processes, as well as the covering and base plates, were removed from all
19
individual vertebrae (final height in each case 15 mm). Osteodensitometric measurements were
20
therefore limited to the central, spongious area of the vertebral bodies. A rectangular region of interest
21
with a defined size of 9 x 11 mm served as measuring field (Fig. 1 (a)). The high density areas of the
22
injected cement were excluded from quantification.
23
2.4 Micro computed tomography (micro-CT)
24
For the acquisition of 3-D images, an X-RAY WorX 225kV tube micro-CT system (X-RAY WorX
25
GmbH, Garbsen, Germany) with a flat panel detector was used (PerkinElmer 1621; CsJ as scintillator;
26
2048 × 2048 pixel; PerkinElmer, Waltham, USA). The following settings were used: 100 kV, 380 μA, 7 Page 7 of 27
1
and a pre-filter of 0.5 mm copper. The shadow images had a size of 1951 × 1813 pixels, and 3600
2
images were recorded for a full 360° turn (duration 6 s; average of 3 × 2 s). A total of 8 frozen samples
3
were analyzed under dry ice within 6 h, resulting in a voxel size of 66.6 μm after reconstruction with
4
the original Feldkamp code (Fig. 2).
5
Quantitative analysis of the micro-CT data was carried out using the 3-D software VGSTUDIO MAX
6
2.2 (Volume Graphics GmbH, Heidelberg, Germany) and applying half-cylinders with a radius of 2.5,
7
3, 3.5, 4, 4.5, and 5 mm (Fig. 3 (a) and (b)). The longitudinal axis and length of the half cylinders for
8
each individual vertebral body were defined on the basis of the respective parameters of the drill
9
channel. The exact 3-D position of the half cylinders (e.g., rotation and depth) was chosen to exclude
10
cortical bone. Bone volume (BV) and total volume (TV) were separately determined by global
11
threshold determination in the drill channel (maximal radius of 2.5 mm) and adjacent half cylinder
12
segments (principle of onion shell). For this purpose, the respective maxima of grey values for soft
13
tissue, bone tissue, and cement were determined and the means between the maxima were used as a
14
threshold for the volume determination of the individual components. The bone volume/total volume
15
(BV/TV) was subsequently calculated.
16
2.5 Histology and histomorphometry
17
After cutting the lumbar vertebral bodies into two parts directly along the axis of the cement injection
18
channel, the analyses were carried out using two different types of histological sections: i) decalcified
19
paraffin sections stained by hematoxylin-eosin [44]; or ii) plastic-embedded sections obtained by
20
fixation in acetone and dehydration in ascending alcohol series without demineralization. Static
21
histomorphometry of the lumbar vertebral bodies (L1-L4) was performed according to published
22
procedures [45–47]. For this purpose, the samples were embedded in Technovit 9100 according to the
23
instructions of the supplier (Heraeus Kulzer, Wehrheim, Germany; [48]). Sections were then cut and
24
ground to a thickness of approx. 7 μm. For static histomorphometry, the sections were stained with
25
trichrome stain according to Masson-Goldner [44].
26
Static histomorphometry of each individual vertebral body in the immediate vicinity of the injection
27
channel was performed using a standard microscope (Axiovert 200 M, Carl Zeiss Microimaging 8 Page 8 of 27
1
GmbH, Oberkochen, Germany) with a 200-fold magnification, and a Merz counting reticule [47, 49].
2
The following 7 parameters were calculated according to the recommendations of the International
3
Committee of the American Society for Bone and Mineral Research (ASBMR) [45, 46]: Bone
4
volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), osteoid
5
volume (OV/BV), osteoid surface (OS/BS), osteoid thickness (O.Th), and eroded Surface (ES/BS).
6
Detailed information on the calculation of the parameters can be found in the supplementary data
7
(including Fig. S2).
8
2.6 Biomechanical testing
9
Biomechanical compressive strength measurements were conducted using a universal material testing
10
machine (FPG 7/20-010™; Kögel, Leipzig, Germany) and the corresponding software (FRK Quicktest
11
2004.01™). For biomechanical testing, frozen cancellous bone cylinders (10 mm diameter x 15 mm
12
height) were obtained from the central part of the vertebral bodies (L1-L4) using a surgical diamond
13
hollow milling cutter (diameter 10 mm). After a defined thawing time of exactly 30 minutes, samples
14
were semi-confined in 2 semilunar clamps (minimal inner diameter of 10.1 mm; length 9.8 mm) and
15
then compressed along their longitudinal axis until fracturing. This axis was chosen because it
16
represents the main loading axis in humans and is therefore of major interest for future clinical
17
application.
18
2.7 Statistics
19
The data were expressed as means ± standard errors of mean. The Wilcoxon test was used to analyze
20
the results of paired samples (samples from the same animals) for statistically significant differences.
21
Differences between 3 and 9 month animals were analyzed for statistical significance using the Mann-
22
Whitney U test. Non-parametric statistical tests were used in the present study due to a lack of normal
23
distribution of the data in a high percentage of the experimental groups. For all tests, the level of
24
significance was set at p ≤ 0.05. All statistical tests were performed using the Sigmaplot software
25
release 13.0 (Systat Software Inc., Chicago, USA).
26
On the basis of a previous study [43] comparing the effects of an injection of pure CPC to an empty
27
defect, an effect size of 0.7 was calculated, i.e. considerably higher than a medium effect size of 0.5. 9 Page 9 of 27
1
Using this effect size in a two-tailed Wilcoxon signed-rank test (matched pairs) with an α of 0.05 and a
2
power of 0.80, a sample number of 19 was computed (G*Power 3.1.9.2). In order to address a
3
potentially lower difference between pure and fiber-reinforced CPC in the present study, the sample
4
number was raised to n = 20 for each of the 2 time points 3 months and 9 months.
5
10 Page 10 of 27
1
3. Results
2
3.1 Osteodensitometry
3
At 3 and 9 months, the bone mineral density (BMD) in vertebral bodies with an empty defect (L2), as
4
well as in both CPC-treated vertebral bodies (L3 and L4; ± PLGA), was significantly higher compared
5
to the untreated control (L1; Fig. 1b). Statistically significant differences between vertebral bodies
6
treated with either pure CPC (L3) or CPC with PLGA fibers (L4) were not observed (Fig. 1b).
7
Interestingly, at 3 months the vertebral bodies treated with CPC with PLGA fibers (L4) also showed a
8
significant BMD increase compared to the empty defect (L2). In addition, the BMD numerically
9
decreased from 3 to 9 months for all three treated vertebral bodies (L2, L3 and L4), but without
10 11
significant differences between the 3 and 9 months values. 3.2 Micro computed tomography
12
In representative 9-month micro-CT images of untreated controls (L1) and treated vertebral bodies
13
(L2, L3, and L4), empty defects (L2) showed incomplete bone healing and remodeling mainly
14
restricted to the original drill channel (Fig.2 (b)). In contrast, both CPC-treated vertebral bodies (L3
15
and L4; ± PLGA) showed an increased spongiosa density directly adjacent to the edge of the drill
16
channel, but still incomplete bone healing and remodeling (Fig. 2 (c) and (d)).
17
At 3 months, quantitative evaluation of the micro-CT data revealed a significantly increased bone
18
volume/total volume as compared to untreated controls (L1) in vertebral bodies with an empty defect
19
(L2) up to a distance of 1 mm from the original defect (Fig. 3 (c)). For higher distances, this bone
20
volume/total volume matched that of the untreated controls (L1). In contrast, the implantation of pure
21
CPC (L3) or CPC with PLGA fibers (L4) significantly enhanced bone formation up to a distance of at
22
least 2.5 mm. At the high distances of 2 and 2.5 mm, this osteoconductive effect was significantly
23
higher for CPC with PLGA fibers (L4) than for pure CPC (L3; Fig. 3 (c)).
24
At 9 months, there was still a significantly increased bone volume/total volume as compared to
25
untreated controls (L1) in vertebral bodies with an empty defect (L2) up to a distance of 0.5 mm from
26
the original defect (Fig. 3 (d)). Also, the implantation of pure CPC (L3) or CPC with PLGA fibers
27
(L4) still significantly enhanced bone formation around the drill channel, however only up to a 11 Page 11 of 27
1
distance of 1 mm and without statistically significant differences between pure CPC (L3) and CPC
2
with PLGA fibers (L4). The values of bone volume/total volume were generally lower after 9 months
3
compared to 3 months, with significant differences for pure CPC (L3) in the immediate vicinity of the
4
drill channel and for the CPC with PLGA fibers (L4) starting at a distance of 1 mm from the original
5
defect (Fig. 3 (d)).
6
3.3 Histomorphometry
7
In representative 9 month histology images of untreated controls (L1) and treated vertebral bodies (L2,
8
L3 and L4), empty defects (L2) confirmed incomplete healing and remodeling mainly in the area of
9
the original defect (Fig. 4 (d)-(f)) as observed in the micro-CT sections. In contrast, the dense bony
10
layer in the vicinity of the CPC implants (L3 and L4; ± PLGA) was also visible in the histology
11
images (Fig. 4 (g)-(m)). Also, there was a clear formation of bone bridges and islands in the cement
12
region of vertebral bodies treated with either pure CPC or CPC with PLGA fibers (L3 and L4; Fig. 4
13
(h), (i), (l) and (m)). There were no signs of inflammatory infiltration close to or distant from the
14
injection channel in either the CPC (L3) or the CPC with PLGA fiber (L4) group at any time point.
15
At 3 and 9 months, bone volume/total volume and trabecular thickness in all treated vertebral bodies
16
(L2, L3, and L4) were significantly higher than in untreated controls (L1; Fig. 5 (a) and (b)). Also,
17
CPC-treated vertebral bodies (L3 and L4; ± PLGA) mostly showed significantly higher values than
18
empty defects (L2; Fig. 5 (a) and (b)). Notably, after 9 months the bone volume/total volume for CPC
19
with PLGA fibers (L4) was significantly higher than that for pure CPC (L3; Fig. 5 (a)), whereas the
20
trabecular thickness at 3 and 9 months was significantly lower for the CPC with PLGA fibers (L4; Fig.
21
5 (b)).
22
In contrast to the findings for bone volume/total volume and trabecular thickness, the trabecular
23
number for all treated vertebral bodies (L2, L3 and L4) was significantly lower than that for untreated
24
controls (L1); in addition, the trabecular number for pure CPC (L3) was significantly lower than that
25
for empty defects (L2; Fig. 5 (c)). In contrast, the trabecular number for the CPC with PLGA fibers
26
was significantly higher than that for empty defects at 9 months (L2) and significantly higher than that
27
for pure CPC at 3 and 9 months (L3; Fig. 5 (c)). 12 Page 12 of 27
1
At 9 months, the bone formation parameters osteoid volume, osteoid surface and osteoid thickness for
2
the CPC with PLGA fibers (L4) were always numerically higher than those for all other vertebral
3
bodies (L1-L3), with statistically significant differences compared to either untreated vertebral bodies
4
(L1; osteoid surface, osteoid thickness) or vertebral bodies treated with pure CPC (L3; osteoid volume,
5
osteoid surface; Fig. 6 (a)-(c)). In parallel, the bone degradation parameter eroded surface was
6
significantly lower for CPC with PLGA fibers (L4) than that for untreated controls (L1; Fig. 6 (d)).
7
3.4 Biomechanical testing
8
At 3 and 9 months, the biomechanical compression strength for all treated vertebral bodies (L2, L3,
9
and L4) was numerically higher than that for untreated controls (L1; Fig. 7). Notably, at 9 months
10
CPC with PLGA fibers (L4) showed significantly higher values than untreated controls (L1) and
11
empty defects (L2; Fig. 7).
13 Page 13 of 27
1
4. Discussion
2
The main aim of the present study was to investigate the biocompatibility of the PLGA fiber
3
component of the fiber-reinforced, brushite-forming CPC. The results were consistent in indicating the
4
following aspects: i) the PLGA fibers had no negative effect on bone formation at any time point or
5
with any of the analyses; ii) the presence of PLGA fibers even enhanced the osteoconductivity of the
6
CPC and led to significantly or numerically increased parameters of bone formation. The latter was
7
demonstrated by significantly enhanced structural parameters (bone volume/total volume at 3 and/or 9
8
months) and bone formation parameters (osteoid volume and osteoid surface at 9 months). The BMD
9
was also enhanced at 3 and 9 months, albeit not at a statistically significant level; the biomechanical
10
compression strength was enhanced at 9 months; and, finally, bone erosion as illustrated by the degree
11
of eroded surface was also numerically reduced at 3 and 9 months. Thus the novel PLGA fiber-
12
reinforced, brushite-forming CPC is highly biocompatible in a sheep lumbar vertebroplasty model, and
13
its PLGA fiber component appears to even support bone formation. These characteristics are very
14
promising in view of using PLGA fibers for the improvement of mechanical properties of inherently
15
brittle CPC and thus for potential application in the therapy of vertebral fractures in load-bearing areas
16
[27, 29, 50].
17
Concerning the main time period of osteoconductivity of the PLGA fibers, some of the parameters
18
indicated predominantly early effects (i.e., BMD, bone volume/total volume in micro-CT and
19
histomorphometry), whereas all bone formation parameters and the biomechanical properties pointed
20
to later effects. In addition, the trabecular structure of the newly formed bone inside or adjacent to the
21
injection channel at 9 months still appeared rather immature with few, but relatively thick trabeculae
22
(Fig. 4). This may have been even more pronounced after 3 months, as indicated by a significantly
23
higher bone volume/total volume in both micro-CT and histomorphometry, as well as by a higher
24
trabecular thickness but lower trabecular number when comparing the 3 months groups to the 9
25
months groups. These findings are in good agreement with previous reports on the presence of only
26
partially remodeled new bone 12 weeks after implantation of a hydroxyapatite-forming CPC
27
containing 31% (w/w) PLGA microspheres into the mandible of adult minipigs [40], as well as with
28
common knowledge on the temporal dynamics of bone healing and remodeling [51]. The present 14 Page 14 of 27
1
findings indicate on one hand that long-term bone remodeling in the vicinity of the injected CPC with
2
fibers (as in the case of the pure CPC) is still ongoing at 9 months. On the other hand, they show that
3
the osteoconductive effects of CPC and/or PLGA fibers may be transient.
4
The differential effects of the PLGA fibers on the osteoconductivity of the composite CPC likely
5
depend on their resorbability in vivo. Depending on their composition, physical shape/size, %
6
proportion, and connectivity (among other factors), PLGA fibers are rapidly resorbed in vivo, resulting
7
in resorption of PLGA-containing CPC between 10% and 90% at 12 weeks [24, 35, 37, 39]. Some
8
evidence of partial degradation of the CPC with fibers was also observed in the present study, with
9
significantly lower values in comparison to pure CPC at distances of 1.0 and 1.5 mm from the drill
10
channel after 3 months, and directly in the cement cylinder after 9 months (data not shown). This
11
indicates that gradual resorption of the PLGA from the periphery to the drill channel may contribute to
12
the enhanced osteoconductivity of the CPC with fibers.
13
Interestingly, significant osteoconductive effects of both CPC and/or CPC with fibers were observed
14
up to a distance of 2.5 mm from the edge of the injection channel, whereas such ‘remote’ effects were
15
limited to a distance of 1 mm in the case of the empty defects. This is compatible with a high
16
degradability of the brushite-forming CPC used in the present study, as well as with the suggested
17
influence of free calcium and inorganic phosphates on the function of osteoprogenitor cells and
18
osteoblasts, and thus on bone formation [8, 52].
19
In the present study, there were no signs of inflammatory infiltration after the use of CPC or CPC
20
enhanced with PLGA fibers at any time point. This is in clear contrast to some studies showing
21
moderate to strong inflammatory reactions following implantation of PGA or PLGA material into
22
bone [32–34]. In these studies, the extent of the inflammatory reaction appeared to increase with a
23
higher volume or proportion of implanted polymer, either PGA or PLGA. Several different factors are
24
believed to contribute to in situ side effects of degradable polymers, including size, location, time
25
point after implantation, and composition [53, 54]. On the other hand, several studies, including our
26
own, found no evidence of inflammation 12 weeks following bone implantation of CPC/PGA or
27
CPC/PLGA composites with considerable percentages of polymers, showing that the composites can 15 Page 15 of 27
1
be carefully designed to prevent unfavorable tissue reactions [36, 55]. The presence of inflammatory
2
cells at early time points, e.g., 4 weeks, cannot be totally excluded [36, 40], but in the present study –
3
even if present – they did not seem to result in negative long-term effects.
4
In general, vertebroplasty/kyphoplasty of vertebral bodies using PMMA or CPC bears the risk of
5
intravascular leakage potentially leading to pulmonary cement embolism and/or arterial blood pressure
6
alterations [56]. However, in a previous own study there were no in vivo signs of systemic
7
intravascular leakage after the injection of CPC [57]. In addition, local intravascular leakage after in
8
vivo application of CPC was only observed in a very limited percentage of vertebral bodies (approx.
9
10%) versus 53% in the case of a high-viscosity PMMA cement (Kyphon® HV-R®12, Medtronic
10
Inc., Milan, Italy). Finally, both pure and fiber-reinforced CPC ex vivo showed a significantly lower
11
intravertebral extrusion into the bone marrow than the high-viscosity PMMA cement [57]. These
12
findings rather argue for a decreased risk of the new CPC material for intravascular leakage.
13
Limitations of the present study
14
In the current study, pure CPC was injected into L3 and CPC with PLGA fibers was injected into L4.
15
Therefore, systematic inter-group statistical significant difference based on the experimental design
16
cannot be totally excluded. However, there were no statistically significant differences in structural
17
bone parameters between L3 and L4 of young sheep (2-4 years; see supplementary data, Figure S3). It
18
is thus not very likely (although not impossible) that systematic differences between the bone
19
regeneration ability in L3 and L4 have biased the results of the present study.
20
Structural and functional effects of CPC and with PLGA fibers on bone regeneration were tested in
21
aged sheep (6-9 years). These animals showed significantly reduced structural histomorphometric
22
parameter compared to young sheep (see supplementary data, Figure S1 and [42]) and were therefore
23
referred to as osteopenic. However, it is not clear whether the significant enhancement of bone
24
formation by the PLGA-fiber component observed in the present study is completely transferable to
25
human osteoporosis. A first step to answer this question may be the use of an ovine model with
26
induced osteoporosis, which, however, requires long-term and costly preparation of the animals [58].
27 16 Page 16 of 27
1
5. Conclusion
2
The fiber component of a PLGA fiber-reinforced, brushite-forming CPC not only lacked negative or
3
toxic effects, but it significantly enhanced bone formation at the vertebral site of injection. The
4
positive effects of the PLGA fibers on bone formation and the augmentation of the mechanical
5
properties of the resulting CPC [27, 29] may thus allow the development of optimized composite CPC
6
for potential application in vertebroplasty or kyphoplasty in (load-bearing) bone defects. One
7
particular advantage of the present composite CPC for future clinical development may be the usage of
8
2 components already CE certified as bone cement and suture material. Finally, the large animal
9
vertebroplasty model in sheep appeared well-suitable for the testing of vertebral augmentation with
10
resorbable and osteoconductive CPC.
17 Page 17 of 27
1
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Figure captions
2
Figure 1: (a) Control of CPC placement by conventional computer tomography; definition of regions
3
of interests (ROI) for osteodensitometry; (b) bone mineral density (BMD) 3 months and 9 months
4
after surgery (n = 20 each),as determined by osteodensitometry in differently treated lumbar vertebral
5
bodies. * p ≤ 0.05 vs. control (L1); + p ≤ 0.05 vs. empty defect (L2).
6
Figure 2: Representative images from micro computed tomography (micro-CT) of differently treated
7
lumbar vertebral bodies 9 months after surgery: (a) untreated control (L1); (b) empty defect (L2); (c)
8
CPC (L3); (d) CPC with PLGA.
9
Figure 3: Quantitative evaluation of the micro computed tomography (micro-CT) of differently treated
10
lumbar vertebral bodies: (a) and (b) schematic representation of the half-cylinders with a radius of 2.5,
11
3, 3.5, 4, 4.5, and 5 mm used for quantitative evaluation; (c) bone volume/total volume (BV/TV) after
12
3 months; (d) bone volume/total volume (BV/TV) after 9 months. * p ≤ 0.05 vs. control (L1); + p ≤
13
0.05 vs. empty defect (L2); § p ≤ 0.05 vs. CPC (L3); # p ≤ 0.05 vs. 3 months.
14
Figure 4: Representative histology images of differently treated, paraffin-embedded lumbar vertebral
15
bodies 9 months after surgery stained by hematoxylin-eosin: (a-c) untreated control (L1); (d-f) empty
16
defect (L2); (e-i) CPC (L3); (k-m) CPC with PLGA. Inserts in (a, b, d, e, g, h, k, and l) indicate the
17
regions shown in a higher magnifications in (b, c, e, f, h, i, l and m), respectively.
18
Figure 5: Structural bone parameters 3 months and 9 months after surgery (n = 20 each), as determined
19
by static histomorphometry in differently treated lumbar vertebral bodies: (a) bone volume/total
20
volume (BV/TV); (b) trabecular thickness (Tb.Th); (c) trabecular number (Tb.N). * p ≤ 0.05 vs.
21
control (L1); + p ≤ 0.05 vs. empty defect (L2); § p ≤ 0.05 vs. CPC (L3); # p ≤ 0.05 vs. 3 months.
22
Figure 6: Bone formation and erosion parameters 3 months and 9 months after surgery (n = 20 each),
23
as determined by static histomorphometry in differently treated lumbar vertebral bodies: (a) osteoid
24
volume (OV/BV); (b) osteoid surface (OS/BS); (c) osteoid thickness (O.Th); (d) eroded surface
25
(ES/BS). * p ≤ 0.05 vs. control (L1); § p ≤ 0.05 vs. CPC (L3); # p ≤ 0.05 vs. 3 months.
25 Page 25 of 27
1
Figure 7: Biomechanical compressive strength of cancellous bone 3 months and 9 months after
2
surgery (n = 20 each) in differently treated lumbar vertebral bodies. * p ≤ 0.05 vs. control (L1); + p ≤
3
0.05 vs. empty (L2).
4
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