The authors are indebted to Geistlich AG (Wolhusen, CH) and the Osteology foundation (Lucerne, CH) (grant n&#176;18-049) for their support, as well as Global D (Brignais, FR) for providing the screws. A particular thanks goes to Dr B. Schaefer from Geistlich. We are also grateful to Eliane Dubois and Claire Herrmann for their excellent histological processing and their precious advices. Finally, we warmly acknowledge Xavier Belin, Sylvie Roulet and the entire team of Pr Walid Habre, &#8220;experimental surgery Dpt&#8221; ,for their remarkable technical assistance.

In line with Swiss legal requirements, the protocol was approved by an academic committee and supervised by the cantonal and federal veterinary agencies (authorizations n&#176; GE/165/16 and GE/100/18).
1. Specific devices and animals
<ol>
	<li>Cylinders
	<ol>
		<li>Machine cylinders with lateral stabilizing tabs out of PEEK to have inner diameter of 5 mm, outer diameter of 8 mm and a height of 5 mm (Figure 1).</li>
		<li>Machine PEEK caps with a design allowing to clip precisely onto the top of the cylinder (thickness 1 mm).</li>
		<li>Sterilize PEEK cylinders and caps by autoclaving before surgery.</li>
	</ol>
	</li>
	<li>Screws
	<ol>
		<li>Use self-drilling micro screws (made of commercial pure titanium (grade 5)) to fix the cylinders (1.2 mm in diameter, 4 mm in length). Sterilize by autoclaving before surgery.</li>
	</ol>
	</li>
	<li>Animals
	<ol>
		<li>Purchase three-month-old New Zealand white rabbits (male or female), weighing ~2.5 kg each. 
		NOTE: We obtained rabbits by breeding at the University of Geneva.</li>
	</ol>
	</li>
</ol>
2. Surgery
<ol>
	<li>Surgical tray 
	<ol>
		<li>Keep scalpels, scissors, two forceps, periosteal elevator, syringes (1, 2, 5, 50 mL), surgical motor, round surgical burs (0.8 mm diameter), needles, sterile saline, four cylinders, eight screws, and screwdriver ready.</li>
	</ol>
	</li>
	<li>Preclinical treatment
	<ol>
		<li>Acclimate the animals one week prior to surgery.</li>
		<li>Provide a prophylactic antibiotic daily (5&#8211;10 mg/kg by mouth (PO)) starting 2 h before surgery up to 3 days after surgery.</li>
	</ol>
	</li>
	<li>Anesthesia and intubation
	<ol>
		<li>Sedate the animals by intramuscular (IM) injection of ketamine (25 mg/kg, 50 mg/mL, 0.5 mL/kg) + xylazin (3 mg/kg, 20 mg/mL, 0.15 mL/kg). Wait ~20 min for the animals to sleep 
		deeply (complete muscular atony). 
		NOTE: This premedication will allow a simple, fast and painless intubation process. Deep analgesia and anesthesia is induced as described in step 2.3.8.</li>
		<li>Place an intravenous (IV) cannula into the marginal vein from the ear and keep it closed until intubation is completed. 
		NOTE: This IV line will serve to perfuse fentanyl and propofol for deep analgesia and anesthesia, respectively (see step 2.3.8).</li>
		<li>Maintain anesthesia by supplying 5% sevoflurane in pure oxygen until intubation is performed. 
		NOTE: This step is necessary only if the animal shows signs of awakening (eye movements, muscular contractions).</li>
		<li>Anesthetize the trachea locally by spraying 10% lidocaine. Place the rabbit in prone position and maintain its head in vertical extension.</li>
		<li>Slide the first endotracheal tube of small diameter (2.5 mm) into the rabbit trachea until airflow can be heard in the tube. This will open the larynx and facilitate the insertion of the definitive tube.</li>
		<li>Insert a guide (intubation catheter) in the tube to fix the position of the tube into the trachea. Remove the small diameter tube and slide the definitive endotracheal tube (4.9 mm) on the guide.</li>
		<li>Remove the guide and inflate the balloon at the end of the endotracheal tube to seal and block the device into the trachea. The tube will stay in place but it may be secured by using a lace tied around the forehead.&#160;&#160;Immediately ventilate (7 mL/kg, frequency of 40/min) the animal with 3% sevoflurane in pure oxygen.</li>
		<li>Continuously perfuse (ear vein) fentanyl (0.01 mg/mL, 2&#8211;4 mL/h) to induce analgesia, 2&#8211;4 mg/kg of (2%) propofol (20 mg/mL, 4&#8211;8 mL/h) to induce anesthesia, and 4 mL/kg/h of Ringer&#39;s acetate to maintain iso-volumetric conditions.</li>
		<li>Place a rectal temperature probe. Also monitor heart function, temperature and oxygen saturation during the entire process.</li>
		<li>Control the depth of anesthesia by monitoring autonomous breathing; if the animal shows signs of autonomous breathing, dispense a small bolus of propofol and fentanyl.</li>
	</ol>
	</li>
	<li>Site preparation
	<ol>
		<li>Place the rabbit on a heated pad (39 &#176;C)&#160;covered by a mattress pad (to avoid burns) on the surgery table. Shave the scalp.</li>
		<li>Apply a lubricating gel on the eyes to avoid irritation and dryness. Disinfect the site by scrubbing the skin with povidone iodine (10%). Then drape the rabbit with a sterile surgical drape and cut out an access area for the skull.</li>
		<li>Disinfect the surgical site with povidone iodine (10%) for a second time. Apply a lubricating gel on the eyes to avoid irritation and dryness.</li>
		<li>Prepare a draped table (sterile drape) on which to place the complete surgical tray.</li>
	</ol>
	</li>
	<li>Surgical site opening
	<ol>
		<li>Anesthetize locally with a subcutaneous (SC) injection of lidocaine 2% (1 mL)&#160;on the skull.</li>
		<li>Incise through the skin (with a scalpel) along the calvarial sagittal line, from the orbits to the external occipital protuberance (~4 cm in length). Ensure that the periosteum is incised.</li>
		<li>Gently elevate the periosteum (with a periosteal elevator) on both side of the incision. Rinse the site with sterile saline.</li>
	</ol>
	</li>
	<li>Cylinder placement
	<ol>
		<li>Locate the median and coronal sutures on the skull (Figure 2A,B). Note that these anatomical lines form a cross. The cylinders will be placed in each of the quadrants defined by the cross, ensuring that the edge of the cylinder is not over the suture (Figure 2C).</li>
		<li>Place the first cylinder on the left upper quadrant (left frontal bone), and try to lay the device flat. Fix in the position with strong hand pressure and screw a micro-screw, until resistance is felt. Ensure that the screw head is flush with the surface of the cylinder tab.</li>
		<li>Repeat the same procedure on the other tab to fix the cylinder tightly onto the skull. Ensure that the cylinder is hermetically fixed to the bone.</li>
		<li>Repeat the procedure on the right upper quarter (right frontal bone), left lower quarter (left parietal bone) and right lower quarter (right parietal bone).</li>
	</ol>
	</li>
	<li>Bone drilling of 5 intramedullary holes within the area circumscribed by the cylinders (Figure 1)
	<ol>
		<li>Drill an intramedullary hole under saline irrigation (0.8 mm in diameter, ~1 mm in depth) with a round bur on the bone, in the center of the area circumscribed by the cylinder. Ensure that bleeding appears.</li>
		<li>Drill two more intramedullary holes along the axis passing through the two tab screws, at the inner edges of the cylinder. Along the perpendicular axe, drill two more intramedullary holes at the inner edges of the cylinder. Ensure that bleeding appears.</li>
		<li>Repeat the operation within the three other cylinders.</li>
	</ol>
	</li>
	<li>Filling cylinders with material samples and capping (Figure 3)
	<ol>
		<li>Prepare the desired bone substitute material according to manufacturer instructions or material specifications.</li>
		<li>Fill the first cylinder to the brim with the material sample and close the cylinder by fitting the cap. Repeat the process in the 3 other cylinders.</li>
	</ol>
	</li>
	<li>Surgical site closure
	<ol>
		<li>Close the skin above the cylinders with an intermittent non-resorbable suture.</li>
		<li>Apply a sprayable dressing onto the wound.</li>
	</ol>
	</li>
</ol>
3. Post-surgical treatment
<ol>
	<li>Stop analgesia and anesthesia (propofol and fentanyl perfusion arrest) supply and check the recovery of autonomous breathing.</li>
	<li>Stop the ventilation once the animal has recovered autonomous breathing. Maintain the animal under pure oxygen before complete awakening.</li>
	<li>Inject buprenorphine hydrochloride SC (0.02 mg/kg, 0.03 mg/mL, 0.67 mL/kg) and repeat the injection every 6 h for 3 days as post-surgical analgesia.</li>
	<li>Transfer the animal into its usual housing with water and complete feeding.</li>
	<li>Remove the sutures after about 10 days of wound healing.</li>
</ol>

The authors have nothing to disclose.

The model described herein is simple and should be developed quite easily as long as all the steps are followed and the equipment is suitable. As the protocol described is a surgical method, all the steps appear critical and must be followed properly. It is critical to be trained for animal experiments, especially in rabbit handling and anesthesia. Do not hesitate to ask for professional anesthetist and veterinary help. It is critical to insist on the daily visual monitoring of animals before and after suture removal. Ev...

The calvarial model of bone augmentation was developed in the 90&#8217;s with the aim to optimize the concept of guided bone regeneration (GBR) in the oral and craniofacial surgical domain. The basic principle of this model is to grow new bone tissue vertically on top of the cortical part of the skull. To do so, a reactor (e.g., titanium -dome, -cylinder or -cage) is fixed onto the skull to protect the bone regeneration conducted by a graft (e.g., hydrogel, bone substitute, etc.). With the aid of this model, titanium or ceramic cages1,2,3,4,5,6, GBR membranes7,8,9,10, osteogenic factors11,12,13,14,15,16,17, new bone substitutes12,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or the mechanism of neovascularization during the bone regeneration process30 were assessed.
From a translational point of view, the calvarial model represents a one-wall defect that can be compared to a class IV defect in the jaw31. The aim is to grow new bone above a cortical area, without any lateral support from endogenous bone walls. The model is thus extremely stringent and assesses the real potential of vertical osteoconduction over the cortical part of the bone. If the model described herein is primarily dedicated to the assessment of osteoconduction in bone substitutes, osteogenesis and/or osteoinduction may be also assessed, as well as vasculogenesis1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30.
Essentially for ethical, practical and economic reasons, the calvarial model was developed in the rabbit in which the bone metabolism and structure are quite relevant when compared to human32. Of the 30 references cited above, 80% used the rabbit calvarial model1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,17,22,23,26,27,28,29,30,33, thus demonstrating the relevance of this animal model. In 2008, the Busenlechner group transferred the calvarial model to the pig, to allow the comparison of eight bone substitutes simultaneously20 (as compared to two bone substitutes with the rabbit). On the other hand, our group transferred the rabbit calvarial model to sheep. In brief, titanium domes were placed on sheep skulls to characterize the osteoconduction of a new 3D-printed bone substitute. These studies allowed us to develop and master the calvarial model and its analysis16,21.
The last three studies cited16,20,21, together with several other investigations12,17,18,19,22,23,24,26,27,28,29, confirmed the great potential of the calvarial model as a screening and characterization model. However, even though the results obtained were quite satisfactory, they also pointed out some limitations: (1) The use of titanium domes, which prevented X-ray diffusion and in turn live micro-CT use. These could not be removed before histological processing, forcing the researchers to embed the samples in poly(methyl methacrylate) resin (PMMA). The resulting analyses were therefore largely limited to topography. (2) High financial costs especially because of the cost of the animals, and costs related to the logistics, maintenance and the surgery of the animals. (3) Difficulties to obtain ethical approvals for large animals.
A recent study by Polo, et al.26 largely improved the model on the rabbit. Titanium domes were replaced by closable cylinders that could be filled with a constant volume of material. Four of these cylinders were placed on rabbit skulls. At completion, the cylinders could be removed so that biopsies were metal-free, introducing much more flexibility concerning sample processing. The rabbit calvarial model became attractive for simultaneous testing with lower costs, easy animal handling and facilitation of sample processing. Taking advantage of these recent developments, we have further improved the model by replacing titanium with PEEK to produce cylinders, thereby allowing X-ray diffusion and the use of microtomography on live animals.
In this article, we will describe the anesthesia and surgery processes and show examples of outputs that may be obtained using this protocol, i.e., (immuno-) histology, histomorphometry, live and ex vivo microtomography to evaluate the mechanisms of bone regeneration and quantify the new bone synthesis supported by bone substitute materials.

<table>
	<tbody>
		<tr>
			<td>Drugs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxacine Baytril 10%</td>
			<td>Bayer</td>
			<td></td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Bischel</td>
			<td></td>
			<td>For analgesia</td>
		</tr>
		<tr>
			<td>Ketalar 50mg/ml</td>
			<td>Pfizer</td>
			<td></td>
			<td>Ketamine for anesthesia</td>
		</tr>
		<tr>
			<td>Lidohex</td>
			<td>Bichsel</td>
			<td></td>
			<td>Lubricating gel for the eyes</td>
		</tr>
		<tr>
			<td>Opsite</td>
			<td>Smith and Nephew</td>
			<td>66004978</td>
			<td>Sprayable dressing</td>
		</tr>
		<tr>
			<td>Povidone iodine 10%, Betadine</td>
			<td>Mundipharma</td>
			<td></td>
			<td>anti-infective agent</td>
		</tr>
		<tr>
			<td>Propofol 2%</td>
			<td>Braun</td>
			<td>3538710</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Rapidocain 2%</td>
			<td>sintetica</td>
			<td></td>
			<td>Local anesthesia</td>
		</tr>
		<tr>
			<td>Ringer-acetate</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td>Volume compensation</td>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazin for anesthesia</td>
		</tr>
		<tr>
			<td>Sevoflurane 5%</td>
			<td>Abbvie</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Sintetica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temgesic</td>
			<td>Reckitt Benckiser</td>
			<td></td>
			<td>Buprenorphine hydrochloride, analgesia</td>
		</tr>
		<tr>
			<td>Thiopental Inresa</td>
			<td>Ospediala</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Xylocaine 10% spray</td>
			<td>Astra Zeneca</td>
			<td></td>
			<td>For intubation</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fresenius Vial pilot C</td>
			<td>Imexmed</td>
			<td></td>
			<td>Infusion pump</td>
		</tr>
		<tr>
			<td>Heated pad</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suction dominant 50</td>
			<td>Medela</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suction tubing Optimus</td>
			<td>Promedical</td>
			<td>80342.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical motor</td>
			<td>Schick dental</td>
			<td>Qube</td>
			<td>Drilling of intramedullary holes</td>
		</tr>
		<tr>
			<td>Ventilation</td>
			<td>Maquet Servo1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cylinders and caps</td>
			<td>Boutyplast</td>
			<td>Customized</td>
			<td>composition: PEEK (poly ether ether ketone)</td>
		</tr>
		<tr>
			<td>Manual self-retaining shaft</td>
			<td>GlobalD</td>
			<td>ACT1K</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobile handle for self-retaining shaft</td>
			<td>GlobalD</td>
			<td>MTM</td>
			<td></td>
		</tr>
		<tr>
			<td>Self- drilling screws</td>
			<td>GlobalD</td>
			<td>VA1.2KL4</td>
			<td>cross-drive screws composed by Titanium grade5, ISO 5832-3</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical tray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube Shiley diameter 2,5mm</td>
			<td>Covidien</td>
			<td>86233</td>
			<td>For intubation</td>
		</tr>
		<tr>
			<td>Endotracheal tube Shiley diameter 4,9mm</td>
			<td>Covidien</td>
			<td>107-35G</td>
			<td>For intubation</td>
		</tr>
		<tr>
			<td>Ethicon prolene 4-0</td>
			<td>Ehticon</td>
			<td>8581H</td>
			<td>Non-resorbable suture</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Marcel Blanc</td>
			<td>BD027R</td>
			<td>145 mm</td>
		</tr>
		<tr>
			<td>Intubation catheter</td>
			<td>Cook medical</td>
			<td></td>
			<td>Guide for intubation</td>
		</tr>
		<tr>
			<td>Needlle holder</td>
			<td>Marcel Blanc</td>
			<td>BM008R</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles BD Microlance3</td>
			<td>Becton Dickinson</td>
			<td>300300/304622</td>
			<td>26G; 18G</td>
		</tr>
		<tr>
			<td>Periosteal</td>
			<td>HU-Friedy</td>
			<td>P9X</td>
			<td></td>
		</tr>
		<tr>
			<td>Round surgical burs</td>
			<td>Patterson</td>
			<td>78000</td>
			<td>0.8 mm in diameter, Drilling of intramedullary holes</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Swann-Morton</td>
			<td></td>
			<td>n&#176;10 and n&#176;15</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Marcel Blanc</td>
			<td>00657</td>
			<td>180 mm</td>
		</tr>
		<tr>
			<td>Syringes Omnifix</td>
			<td>Braun</td>
			<td>4616057V</td>
			<td>5ml, 10ml and 50ml</td>
		</tr>
		<tr>
			<td>Venflon G22</td>
			<td>Braun</td>
			<td>42690985-01</td>
			<td>Vasofix safety for the ear iv line</td>
		</tr>
	</tbody>
</table>

calvarial model of bone augmentation in rabbit for assessment of bone growth and neovascularization in bone substitution materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

The model described herein is dedicated to the assessment of osteoconduction in bone substitutes. Osteogenesis and-or osteoinduction of bone substitutes either (pre-)cellularized or loaded with bioactive molecules may be also assessed, as well as vasculogenesis1,2,3,4,5,6,<sup class...

Watch this Scientific Journal Video about Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials at JoVE.com

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

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8.1K Views.  University of Geneva, University Clinic of Dental Medicine. Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Video: Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is essential to determine the effect of high ground impact forces on lower-body bone deformation to better understand the mechanisms of bone stress injuries. Conventional strain gauge measurement has been used to study in vivo tibia deformation. This method is associated with limitations including invasiveness of the procedure, involvement of few human subjects, and limited strain data from small bone surface areas. The current study intends to introduce a novel approach to study tibia bone strain under high impact loading conditions. A subject-specific musculoskeletal model was created to represent a healthy male (19 years, 80 kg, 1,800 mm). A flexible finite element tibia model was created based on a computed tomography (CT) scan of the subject&#39;s right tibia. Laboratory motion capture was performed to obtain kinematics and ground reaction forces of drop-landings from different heights (26, 39, 52 cm). Multibody dynamic computer simulations combined with a modal analysis of the flexible tibia were performed to quantify tibia strain during drop-landings. Calculated tibia strain data were in good agreement with previous in vivo studies. It is evident that this non-invasive approach can be applied to study tibia bone strain during high impact activities for a large cohort, which will lead to a better understanding of injury mechanism of tibia stress fractures.

During landing, lower-body bones experience large mechanical loads and are deformed. It is essential to measure bone deformation to better understand the mechanisms of bone stress injuries associated with impacts. A novel approach integrating subject-specific musculoskeletal modeling and finite element analysis is used to measure tibial strain during dynamic movements.

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been fabricated with hydroxyapatite to facilitate cell adherence, proliferation, and osteogenesis. In this study, hybrid nanocomposites were successfully developed using graphene nanoribbons (GNRs) and nanoparticles of hydroxyapatite (nHAPs), that when employed in bioactive scaffolds may potentially improve bone tissue regeneration. These nanostructures can be biocompatible. Here, two approaches were used for preparing the novel materials. In one approach, a co-functionalization strategy was used where nHAP was synthesized and conjugated to GNRs simultaneously, resulting in nanohybrids of nHAP on GNR surfaces (denoted as nHAP/GNR). High-resolution transmission electron microscopy (HRTEM) confirmed that the nHAP/GNR composite is comprised of slender, thin structures of GNRs (maximum length of 1.8 &#181;m) with discrete patches (150-250 nm) of needle-like nHAP (40-50 nm in length). In the other approach, commercially available nHAP was conjugated with GNRs forming GNR-coated nHAP (denoted as GNR/nHAP) (i.e., with an opposite orientation relative to the nHAP/GNR nanohybrid). The nanohybrid formed using the latter method exhibited nHAP nanospheres with a diameter ranging from 50 nm to 70 nm covered with a network of GNRs on the surface. Energy dispersive spectra, elemental mapping, and Fourier transform infrared (FTIR) spectra confirmed the successful integration of nHAP and GNRs in both nanohybrids. Thermogravimetric analysis (TGA) indicated that the loss at elevated heating temperatures due to the presence of GNRs was 0.5% and 0.98% for GNR/nHAP and nHAP/GNR, respectively. The nHAP-GNR nanohybrids with opposite orientations represent significant materials for use in bioactive scaffolds to potentially promote cellular functions for improving bone tissue engineering applications.

Novel nanocomposites of graphene nanoribbons and hydroxyapatite nanoparticles were prepared using solution-phase synthesis. These hybrids when employed in bioactive scaffolds can exhibit potential applications in tissue engineering and bone regeneration.

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been ...

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...