BMP-2 (and partially GDF-5) coating significantly accelerates and augments bone formation close to hydroxyapatite/tricalcium-phosphate/brushite implant cylinders for tibial bone defects in senile, osteopenic sheep.

Bilateral defects (diameter 8 mm) in the medial tibial head of senile, osteopenic female sheep (n = 48; 9.63 ± 0.10 years; mean ± SEM) were treated with hydroxyapatite (HA)/beta-tricalcium phosphate (ß-TCP)/dicalcium phosphate dihydrate (DCPD; brushite) cylinders coated with BMP-2 (25 or 250 micrograms) or growth differentiation factor (GDF)-5 (125 or 1250 micrograms; left side); cylinders without BMP served as controls (right side). Three, 6, and 9 months post-operation (n = 6 each group), bone structure and formation were analyzed in vivo by X-ray and ex vivo by osteodensitometry, histomorphometry, and micro-computed tomography (micro-CT) at 3 and 9 months. Semi-quantitative X-ray evaluation showed significantly increasing bone densities around all implant cylinders over time. High-dose BMP-2-coated cylinders (3 and 9 months) and low-dose GDF-5-coated cylinders (3 and 6 months) demonstrated significantly higher densities than controls (dose-dependent for BMP-2 at 3 months). This was confirmed by osteodensitometry at 9 months for high-dose BMP-2-coated cylinders (and selected GDF-5 groups), and was again dose-dependent for BMP-2. Osteoinduction by BMP-2 was most pronounced in the adjacent bone marrow (dynamic histomorphometry/micro-CT). BMP-2 (and partially GDF-5) significantly increased the bone formation in the vicinity of HA/TCP/DCPD cylinders used to fill tibial bone defects in senile osteopenic sheep and may be suitable for surgical therapy of critical size, non-load-bearing bone defects in cases of failed tibial head fracture or defect healing.

Single Application of Low-Dose, Hydroxyapatite-Bound BMP-2 or GDF-5 Induces Long-Term Bone Formation and Biomechanical Stabilization of a Bone Defect in a Senile Sheep Lumbar Osteopenia Model.

Effects of hydroxyapatite (HA) particles with bone morphogenetic BMP-2 or GDF-5 were compared in sheep lumbar osteopenia; in vitro release in phosphate-buffered saline (PBS) or sheep serum was assessed by ELISA. Lumbar (L) vertebral bone defects (Ø 3.5 mm) were generated in aged, osteopenic female sheep (n = 72; 9.00 ± 0.11 years; mean ± SEM). Treatment was: (a) HA particles (2.5 mg; L5); or (b) particles coated with BMP-2 (1 µg; 10 µg) or GDF-5 (5 µg; 50 µg; L4; all groups n = 6). Untouched vertebrae (L3) served as controls. Three and nine months post-therapy, bone formation was assessed by osteodensitometry, histomorphometry, and biomechanical testing. Cumulative 14-day BMP release was high in serum (76-100%), but max. 1.4% in PBS. In vivo induction of bone formation by HA particles with either growth factor was shown by: (i) significantly increased bone volume, trabecular and cortical thickness (overall increase HA + BMP vs. control close to the injection channel 71%, 110%, and 37%, respectively); (ii) partial significant effects for bone mineral density, bone formation, and compressive strength (increase 17%; 9 months; GDF-5). Treatment effects were not dose-dependent. Combined HA and BMPs (single low-dose) highly augment long-term bone formation and biomechanical stabilization in sheep lumbar osteopenia. Thus, carrier-bound BMP doses 20,000-fold to 1000-fold lower than previously applied appear suitable for spinal fusion/bone regeneration and improved treatment safety.

In Vitro Cartilage Regeneration with a Three-Dimensional Polyglycolic Acid (PGA) Implant in a Bovine Cartilage Punch Model.

Resorbable polyglycolic acid (PGA) chondrocyte grafts are clinically established for human articular cartilage defects. Long-term implant performance was addressed in a standardized in vitro model. PGA implants (+/- bovine chondrocytes) were placed inside cartilage rings punched out of bovine femoral trochleas (outer Ø 6 mm; inner defect Ø 2 mm) and cultured for 84 days (12 weeks). Cartilage/PGA hybrids were subsequently analyzed by histology (hematoxylin/eosin; safranin O), immunohistochemistry (aggrecan, collagens 1 and 2), protein assays, quantitative real-time polymerase chain reactions, and implant push-out force measurements. Cartilage/PGA hybrids remained vital with intact matrix until 12 weeks, limited loss of proteoglycans from "host" cartilage or cartilage-PGA interface, and progressively diminishing release of proteoglycans into the supernatant. By contrast, the collagen 2 content in cartilage and cartilage-PGA interface remained approximately constant during culture (with only little collagen 1). Both implants (+/- cells) displayed implant colonization and progressively increased aggrecan and collagen 2 mRNA, but significantly decreased push-out forces over time. Cell-loaded PGA showed significantly accelerated cell colonization and significantly extended deposition of aggrecan. Augmented chondrogenic differentiation in PGA and cartilage/PGA-interface for up to 84 days suggests initial cartilage regeneration. Due to the PGA resorbability, however, the model exhibits limitations in assessing the "lateral implant bonding".

Performance of Calcium Phosphate Cements in the Augmentation of Sheep Vertebrae-An Ex Vivo Study.

Oil-based calcium phosphate cement (Paste-CPC) shows not only prolonged shelf life and injection times, but also improved cohesion and reproducibility during application, while retaining the advantages of fast setting, mechanical strength, and biocompatibility. In addition, poly(L-lactide-co-glycolide) (PLGA) fiber reinforcement may decrease the risk for local extrusion. Bone defects (diameter 5 mm; depth 15 mm) generated ex vivo in lumbar (L) spines of female Merino sheep (2-4 years) were augmented using: (i) water-based CPC with 10% PLGA fiber reinforcement (L3); (ii) Paste-CPC (L4); or (iii) clinically established polymethylmethacrylate (PMMA) bone cement (L5). Untouched (L1) and empty vertebrae (L2) served as controls. Cement performance was analyzed using micro-computed tomography, histology, and biomechanical testing. Extrusion was comparable for Paste-CPC(-PLGA) and PMMA, but significantly lower for CPC + PLGA. Compressive strength and Young's modulus were similar for Paste-CPC and PMMA, but significantly higher compared to those for empty defects and/or CPC + PLGA. Expectedly, all experimental groups showed significantly or numerically lower compressive strength and Young's modulus than those of untouched controls. Ready-to-use Paste-CPC demonstrates a performance similar to that of PMMA, but improved biomechanics compared to those of water-based CPC + PLGA, expanding the therapeutic arsenal for bone defects. O, significantly lower extrusion of CPC + PLGA fibers into adjacent lumbar spongiosa may help to reduce the risk of local extrusion in spinal surgery.

Biodegradable cement augmentation of gamma nail osteosynthesis reduces migration in pertrochanteric fractures, a biomechanical in vitro study.

BACKGROUND: Cut-out of gamma nail often results from poor primary bone stability, suboptimal reduction (varus) and excentric placement of the head element which may lead to "instability" and frequently requires revision. Various studies have shown that augmentation with polymethylmethacrylate cement increases the primary stability of osteosynthesis. However, it has not yet been widely used in fracture treatment due to certain disadvantages, e.g., the lack of osteointegration, the formation of an interface membrane or the presence of toxic monomers. Few studies show that biodegradable bone cements increase the stability of osteosynthesis in different anatomical regions and therefore could be an alternative to polymethylmethacrylate cement in the treatment of pertrochanteric fractures. METHODS: Two biomechanical situations were simulated using 24 Sawbones (simple and multifragmentary pertrochanteric fractures; AO-classification 31-A1 and 31-A2. Both groups were stabilized using the Gamma3® nailing system with and without biodegradable bone cement. Sawbones underwent the same cyclic loading test, simulating 10.000 gait cycles loading the bones with three times body weight. Migration was determined by comparing computed tomography scans recorded before and after the mechanical testing. The three-dimensional migration of the lag screw was calculated, and the rotation of the head around the longitudinal axis was determined. FINDINGS: Biodegradable cement reduced migration by approximately 35% in 31-A1 fractures (25.4% in 31-A2 fractures) and the rotation of the head around the lag screw by approximately 37% in 31-A1 fractures (17.8%, 31-A2). INTERPRETATION: Use of biodegradable bone cement improved the primary stability of gamma nail osteosynthesis in the biomechanical model.

Thickness of the Stifle Joint Articular Cartilage in Different Large Animal Models of Cartilage Repair and Regeneration.

OBJECTIVE: Regulatory guidelines for preclinical cartilage repair studies suggest large animal models (e.g., sheep, goat, [mini]-pig, or horse) to obtain results representative for humans. However, information about the 3-dimensional thickness of articular cartilage at different implantation sites in these models is limited. DESIGN: To identify the most suitable site for experimental surgery, cartilage thickness at the medial femoral condyle (MFC), lateral femoral condyle (LFC), and trochlea in ovine, caprine, and porcine cadaver stifle joints was systematically measured using hematoxylin-eosin staining of 6 µm paraffin sections and software-based image analysis. RESULTS: Regarding all ventral-dorsal regions of the MFC, goat showed the thickest articular cartilage (maximal mean thickness: 1299 µm), followed by sheep (1096 µm) and mini-pig (604 µm), with the highest values in the most ventral and dorsal regions. Also for the LFC, the most ventral regions showed the thickest cartilage in goat (maximal mean thickness: 1118 µm), followed by sheep (678 µm) and mini-pig (607 µm). Except for the mini-pig, however, the cartilage thickness on the LFC was consistently lower than that on the MFC. The 3 species also differed along the transversal measuring points on the MFC and LFC. In contrast, there were no consistent differences for the regional cartilage thickness of the trochlea among goat and sheep (≥780 µm) and mini-pig (≤500 µm). CONCLUSIONS: Based on their cartilage thickness, experimental defects on goat and sheep MFC may be viable options for preclinical cartilage repair studies, in addition to well-established horse models.

Systematic Postoperative Assessment of a Minimally-Invasive Sheep Model for the Treatment of Osteochondral Defects.

To assess the clinical course of a sheep stifle joint model for osteochondral (OC) defects, medial femoral condyles (MFC) were exposed without patella luxation using medial parapatellar skin (3-4 cm) and deep incisions (2-3 cm). Two defects (7 mm diameter; 10 mm depth; OC punch) were left empty or refilled with osteochondral autologous transplantation cylinders (OATS) and explanted after six weeks. Incision-to-suture time, anesthesia time, and postoperative wound or impairment scores were compared to those in sham-operated animals. Implant performance was assessed by X-ray, micro-computed tomography, histology, and immunohistology (collagens 1, 2; aggrecan). There were no surgery-related infections or patellar luxations. Operation, anesthesia, and time to complete stand were short (0.5, 1.4, and 1.5 h, respectively). The wound trauma score was low (0.4 of maximally 4; day 7). Empty-defect and OATS animals reached an impairment score of 0 significantly later than sham animals (7.4 and 4.0 days, respectively, versus 1.5 days). Empty defects showed incomplete healing and dedifferentiation/heterotopic differentiation; OATS-filled defects displayed advanced bone healing with remaining cartilage gaps and orthotopic expression of bone and cartilage markers. Minimally-invasive, medial parapatellar surgery of OC defects on the sheep MFC allows rapid and low-trauma recovery and appears well-suited for implant testing.

The old sheep: a convenient and suitable model for senile osteopenia.

INTRODUCTION: Existing osteoporosis models in sheep exhibit some disadvantages, e.g., challenging surgical procedures, serious ethical concerns, failure of reliable induction of substantial bone loss, or lack of comparability to the human condition. This study aimed to compare bone morphological and mechanical properties of old and young sheep, and to evaluate the suitability of the old sheep as a model for senile osteopenia. MATERIALS AND METHODS: The lumbar vertebral body L3 of female merino sheep with two age ranges, i.e., old animals (6-10 years; n = 41) and young animals (2-4 years; n = 40), was analyzed concerning its morphological and mechanical properties by bone densitometry, quantitative histomorphometry, and biomechanical testing of the corticalis and/or central spongious region. RESULTS: In comparison with young sheep, old animals showed only marginally diminished bone mineral density of the vertebral bodies, but significantly decreased structural (bone volume, - 15.1%; ventral cortical thickness, - 11.8%; lateral cortical thickness, - 12.2%) and bone formation parameters (osteoid volume, osteoid surface, osteoid thickness, osteoblast surface, all - 100.0%), as well as significantly increased bone erosion (eroded surface, osteoclast surface). This resulted in numerically decreased biomechanical properties (compressive strength; - 6.4%). CONCLUSION: Old sheep may represent a suitable model of senile osteopenia with markedly diminished bone structure and formation, and substantially augmented bone erosion. The underlying physiological aging concept reduces challenging surgical procedures and ethical concerns and, due to complex alteration of different facets of bone turnover, may be well representative of the human condition.

In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement.

Bone regeneration of sheep lumbar osteopenia is promoted by targeted delivery of bone morphogenetic proteins (BMPs) via a biodegradable, brushite-forming calcium-phosphate-cement (CPC) with stabilizing poly(l-lactide-co-glycolide) acid (PLGA) fibers. The present study sought to quantify the release and bioactivity of BMPs from a specific own CPC formulation successfully used in previous in vivo studies. CPC solid bodies with PLGA fibers (0%, 5%, 10%) containing increasing dosages of GDF5, BB-1, and BMP-2 (2 to 1000 µg/mL) were ground and extracted in phosphate-buffered saline (PBS) or pure sheep serum/cell culture medium containing 10% fetal calf serum (FCS; up to 30/31 days). Released BMPs were quantified by ELISA, bioactivity was determined via alkaline phosphatase (ALP) activity after 3-day exposure of different osteogenic cell lines (C2C12; C2C12BRlb with overexpressed BMP-receptor-1b; MCHT-1/26; ATDC-5) and via the influence of the extracts on the expression of osteogenic/chondrogenic genes and proteins in human adipose tissue-derived mesenchymal stem cells (hASCs). There was hardly any BMP release in PBS, whereas in medium + FCS or sheep serum the cumulative release over 30/31 days was 11-34% for GDF5 and 6-17% for BB-1; the release of BMP-2 over 14 days was 25.7%. Addition of 10% PLGA fibers significantly augmented the 14-day release of GDF5 and BMP-2 (to 22.6% and 43.7%, respectively), but not of BB-1 (13.2%). All BMPs proved to be bioactive, as demonstrated by increased ALP activity in several cell lines, with partial enhancement by 10% PLGA fibers, and by a specific, early regulation of osteogenic/chondrogenic genes and proteins in hASCs. Between 10% and 45% of bioactive BMPs were released in vitro from CPC + PLGA fibers over a time period of 14 days, providing a basis for estimating and tailoring therapeutically effective doses for experimental and human in vivo studies.

Comparison of Near-Infrared Spectroscopy with Needle Indentation and Histology for the Determination of Cartilage Thickness in the Large Animal Model Sheep.

The suitability of near-infrared spectroscopy (NIRS) for non-destructive measurement of cartilage thickness was compared with the gold standard needle indentation. A combination of NIRS and biomechanical indentation (NIRS-B) was used to address the influence of varying loads routinely applied for hand-guided NIRS during real-life surgery on the accuracy of NIRS-based thickness prediction. NIRS-B was performed under three different loading conditions in 40 osteochondral cylinders from the load-bearing area of the medial and lateral femur condyle of 20 cadaver joints (left stifle joints; female Merino sheep; 6.1 ± 0.6 years, mean ± standard error of the mean). The cartilage thickness measured by needle indentation within the region analyzed by NIRS-B was then compared with cartilage thickness prediction based on NIRS spectral data using partial least squares regression. NIRS-B repeat measurements yielded highly reproducible values concerning force and absorbance. Separate or combined models for the three loading conditions (the latter simulating load-independent measurements) resulted in models with optimized quality parameters (e.g., coefficients of determination R2 between 92.3 and 94.7) and a prediction accuracy of < 0.1 mm. NIRS appears well suited to determine cartilage thickness (possibly in a hand-guided, load-independent fashion), as shown by high reproducibility in repeat measurements and excellent reliability compared with tissue-destructive needle indentation. This may provide the basis for non-destructive, intra-operative assessment of cartilage status quo and fine-tuning of repair procedures.

In Vitro Analysis of Cartilage Regeneration Using a Collagen Type I Hydrogel (CaReS) in the Bovine Cartilage Punch Model.

OBJECTIVE: Limitations of matrix-assisted autologous chondrocyte implantation to regenerate functional hyaline cartilage demand a better understanding of the underlying cellular/molecular processes. Thus, the regenerative capacity of a clinically approved hydrogel collagen type I implant was tested in a standardized bovine cartilage punch model. METHODS: Cartilage rings (outer diameter 6 mm; inner defect diameter 2 mm) were prepared from the bovine trochlear groove. Collagen implants (± bovine chondrocytes) were placed inside the cartilage rings and cultured up to 12 weeks. Cartilage-implant constructs were analyzed by histology (hematoxylin/eosin; safranin O), immunohistology (aggrecan, collagens 1 and 2), and for protein content, RNA expression, and implant push-out force. RESULTS: Cartilage-implant constructs revealed vital morphology, preserved matrix integrity throughout culture, progressive, but slight proteoglycan loss from the "host" cartilage or its surface and decreasing proteoglycan release into the culture supernatant. In contrast, collagen 2 and 1 content of cartilage and cartilage-implant interface was approximately constant over time. Cell-free and cell-loaded implants showed (1) cell migration onto/into the implant, (2) progressive deposition of aggrecan and constant levels of collagens 1 and 2, (3) progressively increased mRNA levels for aggrecan and collagen 2, and (4) significantly augmented push-out forces over time. Cell-loaded implants displayed a significantly earlier and more long-lasting deposition of aggrecan, as well as tendentially higher push-out forces. CONCLUSION: Preserved tissue integrity and progressively increasing cartilage differentiation and push-out forces for up to 12 weeks of cultivation suggest initial cartilage regeneration and lateral bonding of the implant in this in vitro model for cartilage replacement materials.

The GDF5 mutant BB-1 enhances the bone formation induced by an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Targeted delivery of osteoinductive bone morphogenetic proteins (eg, GDF5) in bioresorbable calcium phosphate cement (CPC), potentially suitable for vertebroplasty and kyphoplasty of osteoporotic vertebral fractures, may be required to counteract augmented local bone catabolism and to support complete bone regeneration. The biologically optimized GDF5 mutant BB-1 may represent an attractive drug candidate for this purpose. PURPOSE: The aim of the current study was to test an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming CPC containing low-dose BB-1 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and were filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BB-1 (L5; CPC+fibers+BB-1; 5, 100, and 500 µg BB-1; n=6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BB-1) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (BV/TV) (assessed by micro-CT and histomorphometry), trabecular thickness (Tb.Th), and trabecular number (Tb.N); (3) bone formation, that is, osteoid volume/bone volume (OV/BV), osteoid surface/bone surface (OS/BS), osteoid thickness, mineralizing surface/bone surface (MS/BS), mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BB-1 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BB-1, additional significant effects of BB-1 were demonstrated for BMD, bone structure (BV/TV, Tb.Th, and Tb.N), and bone formation (OS/BS and MS/BS). The BB-1 effects on bone formation at 3 and 9 months were dose dependent, with 100 µg as the potentially optimal dosage. CONCLUSIONS: BB-1 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as 100 µg BB-1 was sufficient to augment middle- to long-term bone formation. A CPC containing the novel GDF5 mutant BB-1 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty and kyphoplasty.

Low-dose BMP-2 is sufficient to enhance the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Bioresorbable calcium phosphate cement (CPC) may be suitable for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures. However, additional targeted delivery of osteoinductive bone morphogenetic proteins (BMPs) in the CPC may be required to counteract the augmented local bone catabolism and support complete bone regeneration. PURPOSE: This study aimed at testing an injectable, poly (l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing low-dose bone morphogenetic protein BMP-2 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BMP-2 (L5; CPC+fibers+BMP-2; 1, 5, 100, and 500 µg BMP-2; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BMP-2) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography [micro-CT] and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralizing surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BMP-2 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BMP-2, additional significant effects of BMP-2 were demonstrated for bone structure (bone volume/total volume, trabecular thickness, trabecular number) and formation (osteoid surface/bone surface and mineralizing surface/bone surface), as well as for the compressive strength. The BMP-2 effects on bone formation at 3 and 9 months were dose-dependent, with 5-100 µg as the optimal dosage. CONCLUSIONS: BMP-2 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as ≤100 µg BMP-2 was sufficient to augment middle to long-term bone formation. The novel CPC+BMP-2 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty/kyphoplasty.

GDF5 significantly augments the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Biodegradable calcium phosphate cement (CPC) represents a promising option for the surgical treatment of osteoporotic vertebral fractures. Because of augmented local bone catabolism, however, additional targeted delivery of bone morphogenetic proteins with the CPC may be needed to promote rapid and complete bone regeneration. PURPOSE: In the present study, an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing the bone morphogenetic protein GDF5 was tested in a sheep lumbar osteopenia model. STUDY DESIGN/SETTING: This is a prospective experimental animal study. METHODS: Defined bone defects (diameter 5 mm) were placed in aged, osteopenic female sheep. Defects were treated with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of GDF5 (L5; CPC+fibers+GDF5; 1, 5, 100, and 500 µg GDF5; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months postoperation, structural and functional effects of the CPC (±GDF5) were assessed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralized surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), both CPC+fibers (L4) and CPC+fibers+GDF5 (L5) numerically or significantly improved all parameters of bone formation, bone resorption, and bone structure. These significant effects were observed both at 3 and 9 months, but for some parameters they were less pronounced at 9 months. Compared with CPC without GDF5, additional significant effects of CPC with GDF5 were demonstrated for BMD and parameters of bone formation and structure (bone volume/total volume, trabecular thickness, and trabecular number, as well as mineralized surface/bone surface). The GDF5 effects were dose-dependent (predominantly in the 5-100 µg range) at 3 and 9 months. CONCLUSIONS: GDF5 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. The results indicated that a local dose as low as ≤100 µg GDF5 may be sufficient to augment middle to long-term bone formation. The novel CPC+GDF5 combination may thus qualify as an alternative to the bioinert, supraphysiologically stiff poly(methyl methacrylate) cement currently applied for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures.

Enhanced bone formation in sheep vertebral bodies after minimally invasive treatment with a novel, PLGA fiber-reinforced brushite cement.

BACKGROUND CONTEXT: Injectable, brushite-forming calcium phosphate cements (CPC) show potential for bone replacement, but they exhibit low mechanical strength. This study tested a CPC reinforced with poly(l-lactide-co-glycolide) acid (PLGA) fibers in a minimally invasive, sheep lumbar vertebroplasty model. PURPOSE: The study aimed to test the in vivo biocompatibility and osteogenic potential of a PLGA fiber-reinforced, brushite-forming CPC in a sheep large animal model. STUDY DESIGN/SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter: 5 mm) were placed in aged, osteopenic female sheep, and left empty (L2) or injected with pure CPC (L3) or PLGA fiber-reinforced CPC (L4; fiber diameter: 25 µm; length: 1 mm; 10% [wt/wt]). Three and 9 months postoperation (n=20 each), the structural and functional CPC effects on bone regeneration were documented ex vivo by osteodensitometry, histomorphometry, micro-computed tomography (micro-CT), and biomechanical testing. RESULTS: Addition of PLGA fibers enhanced CPC osteoconductivity and augmented bone formation. This was demonstrated by (1) significantly enhanced structural (bone volume/total volume, shown by micro-CT and histomorphometry; 3 or 9 months) and bone formation parameters (osteoid volume and osteoid surface; 9 months); (2) numerically enhanced bone mineral density (3 and 9 months) and biomechanical compression strength (9 months); and (3) numerically decreased bone erosion (eroded surface; 3 and 9 months). CONCLUSIONS: The PLGA fiber-reinforced CPC is highly biocompatible and its PLGA fiber component enhanced bone formation. Also, PLGA fibers improve the mechanical properties of brittle CPC, with potential applicability in load-bearing areas.

Decreased extrusion of calcium phosphate cement versus high viscosity PMMA cement into spongious bone marrow-an ex vivo and in vivo study in sheep vertebrae.

BACKGROUND CONTEXT: Vertebroplasty or kyphoplasty of osteoporotic vertebral fractures bears the risk of pulmonary cement embolism (3.5%-23%) caused by leakage of commonly applied acrylic polymethylmethacrylate (PMMA) cement to spongious bone marrow or outside of the vertebrae. Ultraviscous cement and specific augmentation systems have been developed to reduce such adverse effects. Rapidly setting, resorbable, physiological calcium phosphate cement (CPC) may also represent a suitable alternative. PURPOSE: This study aimed to compare the intravertebral extrusion of CPC and PMMA cement in an ex vivo and in vivo study in sheep. STUDY DESIGN/SETTING: A prospective experimental animal study was carried out. METHODS: Defects (diameter 5 mm; 15 mm depth) were created by a ventrolateral percutaneous approach in lumbar vertebrae of female Merino sheep (2-4 years) either ex vivo (n=17) or in vivo (n=6), and injected with: (1) CPC (L3); (2) CPC reinforced with 10% poly(l-lactide-co-glycolide) (PLGA) fibers (L4); or (3) PMMA cement (L5; Kyphon HV-R). Controls were untouched (L1) or empty defects (L2). The effects of the cement injections were assessed in vivo by blood gas analysis and ex vivo by computed tomography (CT), micro-CT (voxel size: 67 µm), histology, and biomechanical testing. RESULTS: Following ex vivo injection, micro-CT documented significantly increased extrusion of PMMA cement in comparison to CPC (+/- fibers) starting at a distance of 1 mm from the edge of the defect (confirmed by histology); this was also demonstrated by micro-CT following in vivo cement injection. In addition, blood gas analysis showed consistently significantly lower values for the fraction of oxygenized hemoglobin/total hemoglobin (FO2Hb) in the arterial blood until 25 minutes following injection of the PMMA cement (p ≤ .05 vs. CPC; 7, 15 minutes). Biomechanical testing following ex vivo injection showed significantly lower compressive strength and Young modulus than untouched controls for the empty defect (40% and 34% reduction, respectively) and all three cement-injected defects (21%-27% and 29%-32% reduction, respectively), without significant differences among the cements. CONCLUSIONS: Because of comparable compressive strength, but significantly lower cement extrusion into spongious bone marrow than PMMA cement, physiological CPC (+/- PLGA fibers) may represent an attractive alternative to PMMA for vertebroplasty or kyphoplasty of osteoporotic vertebral fractures to reduce the frequency or severity of adverse effects.

First-time systematic postoperative clinical assessment of a minimally invasive approach for lumbar ventrolateral vertebroplasty in the large animal model sheep.

BACKGROUND CONTEXT: Large animal models are highly recommended for meaningful preclinical studies, including the optimization of cement augmentation for vertebral body defects by vertebroplasty/kyphoplasty. PURPOSE: The aim of this study was to perform a systematic characterization of a strictly minimally invasive in vivo large animal model for lumbar ventrolateral vertebroplasty. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Lumbar defects (diameter 5 mm; depth approximately 14 mm) were created by a ventrolateral percutaneous approach in aged, osteopenic, female sheep (40 Merino sheep; 6-9 years; 68-110 kg). L1 remained untouched, L2 was left with an empty defect, and L3 carried a defect injected with a brushite-forming calcium phosphate cement (CPC). Trauma/functional impairment, surgical techniques (including drill sleeve and working canula with stop), reproducibility, bone defects, cement filling, and functional cement augmentation were documented by intraoperative incision-to-suture time and X-ray, postoperative trauma/impairment scores, and ex vivo osteodensitometry, microcomputed tomography (CT), histology, static/fluorescence histomorphometry, and biomechanical testing. RESULTS: Minimally invasive vertebroplasty resulted in short operation times (28±2 minutes; mean±standard error of the mean) and X-ray exposure (1.59±0.12 minutes), very limited local trauma (score 0.00±0.00 at 24 hours), short postoperative recovery (2.95±0.29 hours), and rapid decrease of the postoperative impairment score to 0 (3.28±0.36 hours). Reproducible defect creation and cement filling were documented by intraoperative X-ray and ex vivo conventional/micro-CT. Vertebral cement augmentation and osteoconductivity of the CPC was verified by osteodensitometry (CPC>control), micro-CT (CPC>control and empty defect), histology/static histomorphometry (CPC>control and empty defect), fluorescence histomorphometry (CPC>control; all p<.05 for 3 and 9 months), and compressive strength measurements (CPC numerically higher than control; 102% for 3 months and 110% for 9 months). CONCLUSIONS: This first-time systematic clinical assessment of a minimally invasive, ventrolateral, lumbar vertebroplasty model in aged, osteopenic sheep resulted in short operation times, rapid postoperative recovery, and high experimental reproducibility. This model represents an optimal basis for standardized evaluation of future studies on vertebral augmentation with resorbable and osteoconductive CPC.