The CAM assay for human bone regeneration evaluation: B l a n k … · 2017. 5. 12. · Inés...
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Inés Moreno-Jimenez, Gry Hulsart Billström, Janos M. Kanczler, Stuart A. Lanham, Jon I. Dawson, Nicholas D. Evans and Richard OC. Oreffo The CAM assay for human bone regeneration evaluation: The potential of Laponite Clay gel for growth factor delivery ex vivo Faculty of Medicine, Southampton General Hospital [email protected] Introduction 1. To study the potential of the CAM to culture human bone tissue and providing a surrogate blood supply. 2. To develop a method to examine bone formation using the human-avian replacement model. 3. To evaluate the effect of Laponite as a hydrogel vehicle to deliver growth factors and enhance bone formation. Conclusions The current study has demonstrated the validity of the CAM as an ex vivo model to culture human bone and study bone regeneration. The combination of this ex vivo model with the use of high resolution computed tomography and histological analysis provides a powerful method to study the effect of novel constructs in bone tissue engineering. In summary, the main conclusions of this study are: 1. The CAM is able to perfuse the human bone tissue with rapidly growing blood vessels and hence a context for bone remodelling. 2. Implantation of the human bone cylinders on the CAM results in significant bone formation 3. Laponite modulates the deposition of mineralized tissue in the bone cylinders. 4. The addition of BMP2 resulted in the formation of mesenchymal cell condensations (Sox9+) from avian origin and extracellular matrix deposition. ACKNOWLEDGEMENTS: The authors would like to thank the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) for their financial support. Methods Results A B CAM-implanted bone cylinder In vitro cultured bone cylinder D TB TB BM BM # # CAM Figure 1: The CAM integrates with the human bone cylinder providing surrogate blood supply. Bone cylinders were cultured in vitro or CAM-implanted for 7 days. Paraffin sections were stained for Alcian Blue (proteoglycans) and Sirius Red (collagen). # indicates the defect region. Solid arrows indicate avian blood vessels carrying nucleated erythrocytes. Dashed arrows indicate enucleated erythrocytes of human blood vessels. Figure 2: Bone volume significant increase of human bone cylinders following CAM-implantation. Bone cylinders were cultured in vitro, CAM-implanted for 7 days, or maintained at 4 ˚C as control. Each cylinder was μCT-scanned before and after under the same X-ray settings to quantify the relative bone volume change. Data representative of osteoporotic (n=3) and osteoarthritic (n=3) patients. Error bars indicate mean ±S D, p<0.05. CAM-implanted in vitro control -10 0 10 20 Bone volume change (%) ** CAM-implanted in vitro control -10 0 10 20 Bone volume change (%) *** Osteoarthritic patient Osteoporotic patient Cathepsin K Masson’s Trichrome In vitro cultured bone cylinder CAM-implanted bone cylinder TB TB TB TB TB TB TB # # TB Figure 3: CAM implantation of bone cylinders results in osteoclast-mediated bone remodelling. Bone cylinders were cultured in vitro or CAM-implanted for 7 days. Consecutive paraffin sections were stained for modified Masson´s Trichrome (mineralised bone orange-pink, osteoid blue) and Cathepsin K immunostaining as osteoclast acitvity marker. # indicates the defect region. Solid arrows indicate Cathepsin K positive immunostaining. 2) The core of the bone cylinders was left empty (Blank) or perfused with Laponite, Laponite + VEGF or Laponite + BMP2. C Pre incubation μCT scan Post incubation μCT scan Bone cylinder Blank Laponite +/- VEGF +/- BMP2 Day 10 Egg window created 21 days incubation Egg shell window CAM visualization Bone cylinder grafting Window sealing CAM Bone cylinder Day 0 Egg fertilization Day 18 Harvest Fresh human femoral head Histological analysis 1 2 3 Figure 5: CAM-derived mesenchymal cell condensations and matrix deposition following BMP2 delivery on CAM- implanted bone cylinders. Bone cylinders were perfused with Laponite+BMP2 and CAM-implanted for 7 days. Consecutive paraffin sections were stained for modified Alcian Blue (proteoglycans) and Sirius Red (collagen) (A-B), Sox9 (C) GFP immunostaining to detect the cells from avian origin (D) and collagen type II (E). # indicates the defect region. Solid arrows indicate mesenchymal cell condensations between human and avian tissue. . Figure 4: CAM-implanted bone cylinders allow detecting bone volume changes between treatments, but not in vitro culture. Bone cylinders were perfused with Laponite, Laponite +VEGF/BMP2 or Blank, and cultured in vitro or CAM-implanted for 7 days. Bone volume was quantified before and after 7 days incubation and the relative change was calculated for individual cylinders. Error bars indicate mean ± SD, p<0.05. One-way ANOVA with Tukey´s post hoc test. GFP Sox 9 Col type II C A B D E # TB TB TB CAM CAM CAM-implanted bone cylinder In vitro cultured bone cylinder Bone fractures require the development of novel biomaterials which harness both skeletal regeneration and stable blood vessel supply 1 . In particular, injectable hydrogels have gained attention in tissue engineering for their similarities to natural extracellular matrix and their minimally invasive delivery. In the present study we have examined a novel, self-assembling, high-adsorptive clay hydrogel (Laponite) to deliver VEGF or BMP2 and trigger bone formation 2 . Following in vitro studies, it is essential to test biomaterials in the context of full animal physiology before the constructs reach the clinic. In vivo models are then required which typically involve invasive procedure in murine hosts. Thus, less sentient alternative in vivo models should be used when available 3 . In this study, the chorioallantoic membrane of the chick embryo (CAM assay) 4 was used used to cultivate freshly isolated human bone thereby providing an alternative in vivo model using clinically relevant samples. Hence, our aims and objectives are: 1. Kanczler J.M & Oreffo R. O. C. Osteogenesis and angiogenesis: The potential for engineering bone. Eur. Cell. Mater. 15, 100-114 (2008) 2. Dawson, J. I et al .Clay gels for the delivery of regenerative microenvironment. Adv Mater. 23, 3304-8 (2011) 3. Russell, W.M.S. and Burch, R.L., The Principles of Humane Experimental Technique, Methuen, London (1959). 4. Nowak-Sliwinska. The chicken chorioallantoic membrane model in biology, medicine and bioengineering. Angiogenesis; 4-, 25-29 (2014). REFERENCES: 1) Human femoral heads were collected after surgery to freshly isolate bone cylinders. A 2mm drill defect was introduced on their core to mimic the injury area. All bone cylinders were μCT- scanned individually (18μm resolution). 3) Bone cylinders were implanted on the GFP-CAM for 7 days or cultured in vitro in basal media without serum. Post-incubation μCT-scanning was performed and bone cylinders were processed for histology. -10 0 10 20 Bone volume change (%) * Lap Lap VEGF Blank M68 OA CAM-implanted -10 0 10 20 Bone volume change (%) M68 OA in vitro Lap Lap VEGF Blank -10 0 10 20 Bone volume change (%) M64 OA in vitro Lap Blank Lap BMP2 -10 0 10 20 Bone volume change (%) Lap Lap BMP2 Blank F81 NOF# CAM-implanted
Transcript of The CAM assay for human bone regeneration evaluation: B l a n k … · 2017. 5. 12. · Inés...
Inés Moreno-Jimenez, Gry Hulsart Billström, Janos M. Kanczler, Stuart A. Lanham, Jon I. Dawson, Nicholas D. Evans and Richard OC. Oreffo
The CAM assay for human bone regeneration evaluation: The potential of Laponite Clay gel for growth factor delivery ex vivo
Faculty of Medicine, Southampton General Hospital [email protected]
Introduction
1. To study the potential of the CAM to culture human bone tissue and providing a surrogate blood supply.
2. To develop a method to examine bone formation using the human-avian replacement model.
3. To evaluate the effect of Laponite as a hydrogel vehicle to deliver growth factors and enhance bone formation.
Conclusions The current study has demonstrated the validity of the CAM as an ex vivo model to culture human bone and study bone regeneration. The combination of this ex vivo model with the use of high resolution computed tomography and histological analysis provides a powerful method to study the effect of novel constructs in bone tissue engineering. In summary, the main conclusions of this study are: 1. The CAM is able to perfuse the human bone tissue with rapidly growing blood vessels and hence a context for bone remodelling.
2. Implantation of the human bone cylinders on the CAM results in significant bone formation
3. Laponite modulates the deposition of mineralized tissue in the bone cylinders.
4. The addition of BMP2 resulted in the formation of mesenchymal cell condensations (Sox9+) from avian origin and extracellular matrix deposition.
ACKNOWLEDGEMENTS:
The authors would like to thank the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) for their financial support.
Methods
Results
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Figure 1: The CAM integrates with the human bone cylinder providing surrogate blood supply. Bone cylinders were cultured in vitro or CAM-implanted for 7 days. Paraffin sections were stained for Alcian Blue (proteoglycans) and Sirius Red (collagen). # indicates the defect region. Solid arrows indicate avian blood vessels carrying nucleated erythrocytes. Dashed arrows indicate enucleated erythrocytes
of human blood vessels.
Figure 2: Bone volume significant increase of human bone cylinders following CAM-implantation. Bone cylinders were cultured in vitro, CAM-implanted for 7 days, or maintained at 4 ˚C as control. Each cylinder was µCT-scanned before and after under the same X-ray settings to quantify the relative bone volume change. Data representative of osteoporotic (n=3) and osteoarthritic (n=3) patients. Error bars indicate mean ±S D, p<0.05.
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Figure 3: CAM implantation of bone cylinders results in osteoclast-mediated bone remodelling. Bone cylinders were cultured in vitro or CAM-implanted for 7 days. Consecutive paraffin sections were stained for modified Masson´s Trichrome (mineralised bone orange-pink, osteoid blue) and Cathepsin K immunostaining as osteoclast acitvity marker. # indicates the defect region. Solid arrows indicate Cathepsin K positive immunostaining.
2) The core of the bone cylinders was left empty (Blank) or perfused with Laponite, Laponite + VEGF or Laponite + BMP2.
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Figure 5: CAM-derived mesenchymal cell condensations and matrix deposition following BMP2 delivery on CAM-implanted bone cylinders. Bone cylinders were perfused with Laponite+BMP2 and CAM-implanted for 7 days. Consecutive paraffin sections were stained for modified Alcian Blue (proteoglycans) and Sirius Red (collagen) (A-B), Sox9 (C) GFP immunostaining to detect the cells from avian origin (D) and collagen type II (E). # indicates the defect region. Solid arrows indicate mesenchymal cell condensations between human and avian tissue. .
Figure 4: CAM-implanted bone cylinders allow detecting bone volume changes between treatments, but not in vitro culture. Bone cylinders were perfused with Laponite, Laponite +VEGF/BMP2 or Blank, and cultured in vitro or CAM-implanted for 7 days. Bone volume was quantified before and after 7 days incubation and the relative change was calculated for individual cylinders. Error bars indicate mean ± SD, p<0.05. One-way ANOVA with Tukey´s post hoc test.
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CAM-implanted bone cylinder In vitro cultured bone cylinder
Bone fractures require the development of novel biomaterials which harness both skeletal regeneration and stable blood vessel supply1 . In particular, injectable hydrogels have gained attention in tissue engineering for their similarities to natural extracellular matrix and their minimally invasive delivery. In the present study we have examined a novel, self-assembling, high-adsorptive clay hydrogel (Laponite) to deliver VEGF or BMP2 and trigger bone formation2. Following in vitro studies, it is essential to test biomaterials in the context of full animal physiology before the constructs reach the clinic. In vivo models are then required which typically involve invasive procedure in murine hosts. Thus, less sentient alternative in vivo models should be used when available3. In this study, the chorioallantoic membrane of the chick embryo (CAM assay)4 was used used to cultivate freshly isolated human bone thereby providing an alternative in vivo model using clinically relevant samples. Hence, our aims and objectives are:
1. Kanczler J.M & Oreffo R. O. C. Osteogenesis and angiogenesis: The potential for engineering bone. Eur. Cell. Mater. 15, 100-114 (2008) 2. Dawson, J. I et al .Clay gels for the delivery of regenerative microenvironment. Adv Mater. 23, 3304-8 (2011) 3. Russell, W.M.S. and Burch, R.L., The Principles of Humane Experimental Technique, Methuen, London (1959). 4. Nowak-Sliwinska. The chicken chorioallantoic membrane model in biology, medicine and bioengineering. Angiogenesis; 4-, 25-29 (2014).
REFERENCES:
1) Human femoral heads were collected after surgery to freshly isolate bone cylinders. A 2mm drill defect was introduced on their core to mimic the injury area. All bone cylinders were µCT-scanned individually (18µm resolution).
3) Bone cylinders were implanted on the GFP-CAM for 7 days or cultured in vitro in basal media without serum. Post-incubation µCT-scanning was performed and bone cylinders were processed for histology.
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