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”

Three step flow focusing enables image-based discrimination and sorting of late stage 1 Haematococcus pluvialis cells.

Label-free and gentle separation of cell stages with desired target properties from mixed stage populations are a major research task in modern biotechnological cultivation process and optimization of micro algae. The reported microfluidic sorter system (MSS) allows the subsequent investigation of separated subpopulations. The implementation of a viability preserving MSS is shown for separation of late stage 1 Haematococcus pluvialis (HP) cells form a mixed stage population. The MSS combines a three-step flow focusing unit for aligning the cells in single file transportation mode at the center of the microfluidic channel with a pure hydrodynamic sorter structure for cell sorting. Lateral displacement of the cells into one of the two outlet channels is generated by piezo-actuated pump chambers. In-line decision making for sorting is based on a user-definable set of image features and properties. The reported MSS significantly increased the purity of target cells in the sorted population (94%) in comparison to the initial mixed stage population (19%)

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

Abstract 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 (β-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. Graphical Abstrac