This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Semiautomated longitudinal micro–computed tomography–based quantitative structural analysis of bone regeneration in osteoporotic rats
Dan Gazit
Gazit Lab
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Osteoporosis-related fractures are becoming more common as the world population ages, and they constitute an unmet clinical need. Animal models are essential for the preclinical development of novel therapeutic strategies, such as stem cells, for osteoporosis-related fractures. While a number of animal models exist, this protocol describes a highly reproducible method of generating multiple bone defects in young osteoporotic immunodeficient rats. In addition, we describe a novel longitudinal, semiautomated, micro–computed tomography-based quantitative structural analysis of bone regeneration. This imaging approach can be adapted for a variety of other bone defects models in which longitudinal imaging–based analyses could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis and 2.7 to 4 hours to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.
Keywords
osteoporosis, vertebral fracture, rodent model, micro–computed tomography
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More than 200 million people worldwide suffer from osteoporosis [1]. The underlying pathological decrease in bone mineral density (BMD) and altered bone microarchitecture increase bone fragility and consequently the risk for fractures, disability and mortality [2]. Beyond the personal impact, fragility fractures represent a major and growing socioeconomic burden as the world’s population ages. In this protocol, osteoporosis was induced in young athymic rats by means of ovariectomy (OVX) coupled with 4 months of low calcium diet (LCD). We were the first to show[3] that this protocol induces an efficient, rapid, and irreversible decrease in vertebral trabecular bone volume and BMD, which mimics postmenopausal osteoporosis. The unique selection of athymic rats allows subsequent research into translational therapeutic strategies such as the use of human stem cells without the limitations imposed by the rodent immune system.
Osteoporotic vertebral compression fractures (OVCFs) are the most common fragility fractures. They are associated with significant morbidity and as much as a nine times higher mortality rate [4]. Currently available surgical interventions, such as vertebroplasty and kyphoplasty, were recently found to be no more effective than a sham treatment in clinical trials [5, 6] leaving only pain management available to these patients. Since current OVCF treatments are limited, it is imperative to develop animal models that can replicate the disorder [7-9]. Such animal models could facilitate the development of novel therapies that will translate into clinical practice. Researchers have created a cylindrical void in the center of the vertebral body to mimic OVCFs [10-17]. Since there is no consistency in the literature in terms of defect size, we have defined critical-sized as a defect that does not fully heal without an intervention within 3 months post-op [11, 16].
Bone imaging is a crucial part of evaluation of fractures and bone diseases. Advanced imaging methods were developed for accurate assessment of structural bone changes and regeneration strategies [18]. Among them, microCT (µCT) imaging has emerged as a non-invasive, easy to use and inexpensive method, which provides high-resolution 3D images with exposure to low levels of radiation. µCT imaging has several advantages over other modalities in evaluating osteoporosis patients, as it offers high-resolution 3D bone microarchitecture [19] that could then be analyzed quantitatively. The latter could then be used to compare therapeutic effects of proposed treatments. In this protocol, the vertebral defects were monitored via a sequence of in-vivo µCT scans at set time points for longitudinal assessment of bone repair. The vertebrae were then contoured and separated from the rest of the scan. A commercially available multiple image registration algorithm facilitated the extraction of an anatomically corresponding baseline VOIs to all subsequent time points. This technique provided a highly accurate and straightforward longitudinal 3D µCT analysis that is not user-dependent.
The methods described here for longitudinal assessment of bone repair using in-vivo µCT scans and a registration algorithm could be applied to any bone defect regeneration analysis. The vertebral defect model used here is a convenient model for this application, as its bone structure is unique and can be easily registered to the same anatomical position. However, any bone regeneration could be analyzed under the same conditions by properly separating the same bone of interest throughout the longitudinal scans. It is imperative to have the separated bone samples with the same anatomical features included. The anatomic match obtained by the registration procedure can only occur if the samples include the same anatomical features. The registration will allow the user to apply the exact predefined VOI of the first scan to all remaining time points, resulting in a highly accurate 3D histomorphometric analysis over time. Bone volume density and apparent density of the VOI can be used to assess new bone formation.
The experimental design of this protocol is shown in figure 1. Briefly, six week-old rats with their ovaries surgically removed were purchased and subjected to a special LCD consisting of 0.01% calcium and 0.77% phosphate. After a period of 4 months of LCD, the rats were operated on and a critical-size vertebral defect was drilled in the fourth and fifth lumbar vertebral bodies (L4–L5). Following surgery, the rats were imaged on day 1, week 2, 4, 8 and 12 after defect establishment. Defect margins of day 1 scans were located and reoriented to a standard position and a cylindrical VOI was defined. Subsequent µCT scans (i.e., for weeks 2, 4, 8 and 12) of each rat were automatically registered to standard position defined for the corresponding day 1 scan using commercially available software. Day 1 predefined VOI was applied to registered scans. Bone volume density and apparent density of the VOI were used to assess new bone formation.
• Ovariectomized athymic rats (Harlan Laboratories, Indianapolis, IN).
• Low calcium food (Newco Distributors, Inc., CA)
• Isoflurane (Patterson Veterinary)
• Phosphate Buffered Saline (PBS, Sigma-Aldrich, St. Louis, MO)
• Betadine (Generic)
• Chlorhexidine Gluconate 0.5% (Generic)
• Ethanol, 100% (Ethyl Alcohol 70% (vol/vol), Nedalco)
• Carprofen (Rimadyl, Zoetis, Florham Park, NJ)
• Ethicon mini Dermabond (Ethicon, Somerville, NJ)
• Buprenorphine hydrochloride (BD Pharmaceuticals)
• Anesthetics machine (Vet Tech Solutions)
• Ventilator (Active scavenger unit, Vet Tech Solutions)
• Induction chamber (Kent Scientific)
• Heating Pad (Rothacher Medical)
• Surgical drape (Medline Industries)
• Magnetic fixator retraction system (Fine Science Tools, Inc., CA)
• Microdissection scissors, 3.5 inch (Roboz Surgical Instruments)
• Scissors (Aesculap)
• Forceps, no. 5 (Roboz Surgical Instruments)
• Hemostatic forceps, straight, 3.5 inch (Roboz Surgical Instruments)
• Sterile cotton gauze (Klinion)
• Cotton swabs (sterile; EMRO Medical)
• Syringe, 1 ml (BD Plastipak)
• Needle, 25-gauge (BD Microlance)
• Laminar flow hood
• Thermal Cautery (World Precision Instruments, FL)
• Micro-Drill (Fine Science Tools, CA)
• Trephines for Micro Drill, 2mm diameter (Fine Science Tools, CA)
• 3-0 Vicryl undyed 27" SH taper (Ethicon, Somerville, NJ)
• 4-0 Ethilon black 18" PC-3 conventional cutting (Ethicon, Somerville, NJ)
• Cone-beam in vivo microCT (vivaCT 40, Scanco Medical was used in this protocol)
• Analyze software (Version 11.0; Biomedical Imaging, Mayo Clinic, Rochester, MN)
Induction of osteoporosis
1) Put six week-old athymic ovariectomized rats on a 4 months of low calcium diet (LCD) consisting of 0.01% calcium and 0.77% phosphate. Then, switch back to normal diet. These rats will be referred to as “osteoporotic rats” hereafter.
CAUTION: Research protocols involving the use of animals should be reviewed by the investigator’s institutional ethical review board to avoid any unnecessary discomfort or pain to the animals and to determine whether alternatives exist to animal research. All animal experiments should be performed in accordance with relevant guidelines and regulations of protocols approved by the investigator’s institutional animal research review committee. Personnel should be trained in animal handling.
Vertebral defect model
2) Anesthesia. Place the osteoporotic rat in an induction chamber of an anesthesia machine with a central scavenging system. Induce anesthesia using 5% isoflurane in 100% oxygen and maintain via nose cone at 2-3% isoflurane.
3) Apply toe pinch stimulus to ensure adequate plane of anesthesia. If no response is noted, the procedure can be initiated.
4) Preoperative care. Place the rat in dorsal recumbency on a heating pad (37 °C).
5) CRITICAL STEP: The temperature of the heating pad is important to prevent hypothermia since an anesthetized rat is unable to regulate its body temperature. Stretch the rat limbs using a magnetic fixator retraction system (Fig. 2A)
6) Shave the abdominal area and swab it with Betadine and Chlorhexidine Gluconate 0.5% followed by 70% ethanol.
7) Inject the rat with Carprofen (5 mg/kg BW, SQ) before beginning the surgical procedure.
8) Surgical procedure. Use sterile surgical scissors to cut the skin. Begin the incision 1cm below the xiphoid process and cut through the midline (~5-8 cm) (Fig. 2B).
CAUTION: Avoid using a scalpel for abdominal incisions. Using surgical scissors, as opposed to a scalpel, reduces bleeding and damage to underlying tissues.
CAUTION: All surgical tools must be autoclaved a day before surgery. In case of multiple surgeries, appropriate sterilization must be applied to all surgical tools. Between surgeries, wash the tools and place them in a sonicator bath for 5 minutes. Then, spray the surgical tools with Isopropyl Alcohol 70% and dry them. Next, place them for 20 seconds in a hot bead sterilizer set to 250°C. Allow the tools to cool down for 5 minutes. Now, the surgical tools can be used for the next surgery.
9) Use surgical scissors to make an incision of the aponeurosis through the linea alba to access the abdominal cavity. (Fig. 2C).
10) Use retractors to expose the abdominal cavity (Fig. 2D).
11) Deflect the intestines to the rat’s right to expose the abdominal aorta and the left kidney (Fig. 2E). Palpate the lumbar spine before proceeding to expose it.
CAUTION: To avoid dehydration, use sterile saline soaked gauzes to wrap the internal organs.
12) Use thermocautery to expose the anterior aspect of lumbar vertebral bodies and isolate them from surrounding connective tissue and muscles (Fig. 2F-G).
CAUTION: Use a cotton swab saturated with 3% hydrogen peroxide to remove blood and residual tissue from the vertebrae.
13) Use a sterile drill bit (~2mm in diameter) to drill a 5mm deep bone defect in the center of the vertebral body (Fig. 2H-I).
CRITICAL STEP: Apply minimal pressure to drill through only the ventral cortex and underlying trabecular bone and avoid drilling through the dorsal cortex. Note that the vertebrae of osteoporotic rats are very fragile.
CAUTION: Use a cotton swab to clean the defect and apply pressure to stop bleeding if present.
14) Repeat step 13 on an adjacent vertebra, to create a total of 2 defects per rat (Fig. 2J).
15) Return the intestines to the abdominal cavity.
16) Use a vicryl synthetic absorbable surgical suture (3-0 Vicryl undyed 27" SH taper) in a continuous pattern to suture the aponeurosis (Fig. 2K).
17) Close the skin using a monofilament nylon non-absorbable suture (4-0 Ethilon black 18" PC-3 conventional cutting) in a simple interrupted pattern (Fig. 2L).
18) Apply 100ul of Dermabond on top of the skin sutures and between them to ensure complete closure of the skin.
19) Postoperative care. Inject the rat with warm (37°C) lactated ringer’s solution (1CC/100g BW, SQ) to prevent hypothermia and dehydration.
20) Inject the rat with Buprenorphine (0.5 mg/kg, SQ) for post-op pain relief.
21) After the animal is recovered on the heating pad, return it to its cage.
CAUTION: House the rats individually, in separate cages, to prevent rat-to-rat mutilation of the sutures and wound.
22) Place chow soaked in water in a petri dish on the cage floor for a few days post-op to help the rats reach the food.
23) Administer Carprofen (5 mg/kg BW, SQ) 24 hours post-surgery for pain relief.
24) Remove skin sutures while animal is under 2% isoflurane anesthesia 10-14 days post-op.
MicroCT scanning
25) One day after the surgical procedure, place the osteoporotic rat in an induction chamber of an anesthesia machine with central scavenging system. Induce anesthesia using 5% isoflurane in 100% oxygen and maintain via nose cone at 2-3% isoflurane.
26) Scan the rat using an in-vivo microCT scanner. Repeat scanning for longitudinal analysis of bone regeneration.
CRITICAL STEP: Make sure that all animals are scanned using the same settings (X-ray energy, scanning medium, intensity, voxel size and image resolution) and in a similar orientation. Refer to Bouxtein et al[1] for further explanations and considerations involved in rodent microCT scanning for assessment of bone microstructure.
Vertebral separation
27) Contour the vertebra of interest, as demonstrated in Fig. 3A-I. Make sure to include all parts of the vertebra while excluding parts that belong to adjacent vertebrae.
28) Save the contoured vertebra as a separate file (Fig. 3J-K).
Definition of the VOI for longitudinal quantitative evaluation
29) The following steps depend on whether the scan is from day 1 after surgery (reference vertebra) or from the subsequent time points (target vertebrae).
(A) Reference vertebra
\(i) Z rotation. Measure the angle of the margins using a xy slice from the center of the defect \(Fig. 4A-B). Use the measured angle to rotate the vertebra \(Fig. 4C).
\(ii) X rotation. Measure the angle of the margins using a yz slice from the center of the defect \(Fig. 4D-E). Use the measured angle to rotate the vertebra \(Fig. 4F).
\(iii) Flip. Flip the rotated vertebra by changing the xy plane to the zx plane.
\(iv) VOI definition. Draw a circular contour of the defect using a slice from the center of the defect \(Fig. 6A). Then, copy that contour and paste it on all the slices in the defect \(Fig. 6B). CRITICAL STEP: Since all defects were created using the same procedure, the same number of slices and subsequently total volume (TV) should be analyzed for all samples.
(B) Target vertebra
\(i) Load the DICOM files of both the target and the reference vertebrae to the main window of the “Analyze” software. CRITICAL STEP: To avoid gray scale value changes, define the same output data type as the original DICOM files in the load menu.
\(ii) Registration to reference vertebra. Launch the “3-D Voxel Registration” module, and input the reference vertebra as the “Base Volume” and the target vertebra as the “Match Volume”. Click “Register” to register the vertebrae \(Fig. 6).
\(iii) Save the registered file using the same data type and import it to your microCT environment.
\(iv) Application of VOI. Apply the VOI defined for the reference vertebra to the registered target vertebra. Check that the VOI and defect are concentric. MicroCT analysis
30) Send the VOI for evaluation using your microCT evaluation program.
CRITICAL STEP: Make sure to use the same parameters when analyzing all VOIs. Make sure the threshold is set high enough to omit background noise with minimal loss of bone.
Steps 2-24, Vertebral defect model: 40-50 minutes
Steps 25-26, MicroCT scan: 30-40 minutes
Steps 27-28, Vertebral separation: 20-30 minutes per sample
Step 29, VOI definition: 20-30 minutes per reference vertebra, 10-20 minutes per target vertebra
Step 30, MicroCT analysis: 10-20 minutes
See Figures section for a table giving troubleshooting guidance.
Using this protocol, one can image and quantify the regeneration of modeled osteoporotic vertebral defects across different time points. The anatomic match obtained by the registration procedure allows the analysis of the exact same VOI at all time points. Resulting in a highly accurate longitudinal 3D histomorphometric analysis even when the margins of the original defect are no longer recognizable. We used five time points (Day 1, Week 2, Week 4, Week 8 and week 12) as an example for longitudinal evaluation of bone regeneration (Fig. 7). Regenerated can be evaluated by both qualitative assessment of 2D cross-sections and 3D images (as illustrated in Figure 7A), and quantitative comparison of the bone quantity (BVD) and quality (AD) (Fig. 7B). The following morphometric indices can be determined for newly formed bone in regeneration sites: (i) TV of bone, including new bone and soft-tissue cavities (TV, mm3); (ii) volume of mineralized bone tissue (BV, mm3); (iii) bone volume density, which can also be referred to as the percentage of regeneration for the round critical-size bone defect model (BV/TV); and (iv) bone mineral density (BMD, mg hydroxyapatite per cm3), derived from comparison of X-ray attenuation in the scanned bone sample with that of hydroxyapatite standards.
- Wang, M. L. et al. A rat osteoporotic spine model for the evaluation of bioresorbable bone cements. The spine journal : official journal of the North American Spine Society 7, 466-474, doi:10.1016/j.spinee.2006.06.400 (2007).
- Consensus development conference: prophylaxis and treatment of osteoporosis. The American journal of medicine 90, 107-110 (1991).
- Sheyn, D. et al. PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries. Mol Ther, doi:10.1038/mt.2015.211 (2015).
- Center, J. R. et al. Mortality after all major types of osteoporotic fracture in men and women: an observational study. Lancet 353, 878-882, doi:10.1016/S0140-6736(98)09075-8 (1999).
- Buchbinder, R. et al. A randomized trial of vertebroplasty for painful osteoporotic vertebral fractures. The New England journal of medicine 361, 557-568, doi:10.1056/NEJMoa0900429 (2009).
- Kallmes, D. F. et al. A randomized trial of vertebroplasty for osteoporotic spinal fractures. The New England journal of medicine 361, 569-579, doi:10.1056/NEJMoa0900563 (2009).
- Kado, D. M. et al. Vertebral fractures and mortality in older women: a prospective study. Study of Osteoporotic Fractures Research Group. Arch Intern Med 159, 1215-1220 (1999).
- Silverman, S. L. The clinical consequences of vertebral compression fracture. Bone 13 Suppl 2, S27-31 (1992).
- Ross, P. D. Clinical consequences of vertebral fractures. Am J Med 103, 30S-42S; discussion 42S-43S (1997).
- Vanecek, V. et al. The combination of mesenchymal stem cells and a bone scaffold in the treatment of vertebral body defects. European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society 22, 2777-2786, doi:10.1007/s00586-013-2991-2 (2013).
- Liang, H. et al. Use of a bioactive scaffold for the repair of bone defects in a novel reproducible vertebral body defect model. Bone 47, 197-204, doi:10.1016/j.bone.2010.05.023 (2010).
- Kobayashi, H. et al. Long-term evaluation of a calcium phosphate bone cement with carboxymethyl cellulose in a vertebral defect model. J Biomed Mater Res A 88, 880-888, doi:10.1002/jbm.a.31933 [doi] (2009).
- Fujishiro, T. et al. Histological evaluation of an impacted bone graft substitute composed of a combination of mineralized and demineralized allograft in a sheep vertebral bone defect. J Biomed Mater Res A 82, 538-544, doi:10.1002/jbm.a.31056 [doi] (2007).
- Sheyn, D. et al. Gene-modified adult stem cells regenerate vertebral bone defect in a rat model. Molecular pharmaceutics 8, 1592-1601, doi:10.1021/mp200226c (2011).
- Wang, M. L. et al. Altered bioreactivity and limited osteoconductivity of calcium sulfate-based bone cements in the osteoporotic rat spine. The spine journal : official journal of the North American Spine Society 8, 340-350, doi:10.1016/j.spinee.2007.06.003 (2008).
- Liang, H. et al. A novel strategy of spine defect repair with a degradable bioactive scaffold preloaded with adipose-derived stromal cells. Spine J 14, 445-454, doi:10.1016/j.spinee.2013.09.045 (2014).
- Phillips, F. M. et al. In vivo BMP-7 (OP-1) enhancement of osteoporotic vertebral bodies in an ovine model. The spine journal : official journal of the North American Spine Society 6, 500-506, doi:10.1016/j.spinee.2006.01.014 (2006).
- Geusens, P. et al. High-resolution in vivo imaging of bone and joints: a window to microarchitecture. Nature reviews. Rheumatology 10, 304-313, doi:10.1038/nrrheum.2014.23 (2014).
- Genant, H. K., Engelke, K. & Prevrhal, S. Advanced CT bone imaging in osteoporosis. Rheumatology 47 Suppl 4, iv9-16, doi:10.1093/rheumatology/ken180 (2008).
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Associated Publications
PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries
by Dmitriy Sheyn, Galina Shapiro, Wafa Tawackoli, +16
Molecular Therapy (16 March, 2016)
Method Article
Semiautomated longitudinal micro–computed tomography–based quantitative structural analysis of bone regeneration in osteoporotic rats
Dan Gazit
Gazit Lab
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 01 Apr, 2016
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Osteoporosis-related fractures are becoming more common as the world population ages, and they constitute an unmet clinical need. Animal models are essential for the preclinical development of novel therapeutic strategies, such as stem cells, for osteoporosis-related fractures. While a number of animal models exist, this protocol describes a highly reproducible method of generating multiple bone defects in young osteoporotic immunodeficient rats. In addition, we describe a novel longitudinal, semiautomated, micro–computed tomography-based quantitative structural analysis of bone regeneration. This imaging approach can be adapted for a variety of other bone defects models in which longitudinal imaging–based analyses could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis and 2.7 to 4 hours to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
More than 200 million people worldwide suffer from osteoporosis [1]. The underlying pathological decrease in bone mineral density (BMD) and altered bone microarchitecture increase bone fragility and consequently the risk for fractures, disability and mortality [2]. Beyond the personal impact, fragility fractures represent a major and growing socioeconomic burden as the world’s population ages. In this protocol, osteoporosis was induced in young athymic rats by means of ovariectomy (OVX) coupled with 4 months of low calcium diet (LCD). We were the first to show[3] that this protocol induces an efficient, rapid, and irreversible decrease in vertebral trabecular bone volume and BMD, which mimics postmenopausal osteoporosis. The unique selection of athymic rats allows subsequent research into translational therapeutic strategies such as the use of human stem cells without the limitations imposed by the rodent immune system.
Osteoporotic vertebral compression fractures (OVCFs) are the most common fragility fractures. They are associated with significant morbidity and as much as a nine times higher mortality rate [4]. Currently available surgical interventions, such as vertebroplasty and kyphoplasty, were recently found to be no more effective than a sham treatment in clinical trials [5, 6] leaving only pain management available to these patients. Since current OVCF treatments are limited, it is imperative to develop animal models that can replicate the disorder [7-9]. Such animal models could facilitate the development of novel therapies that will translate into clinical practice. Researchers have created a cylindrical void in the center of the vertebral body to mimic OVCFs [10-17]. Since there is no consistency in the literature in terms of defect size, we have defined critical-sized as a defect that does not fully heal without an intervention within 3 months post-op [11, 16].
Bone imaging is a crucial part of evaluation of fractures and bone diseases. Advanced imaging methods were developed for accurate assessment of structural bone changes and regeneration strategies [18]. Among them, microCT (µCT) imaging has emerged as a non-invasive, easy to use and inexpensive method, which provides high-resolution 3D images with exposure to low levels of radiation. µCT imaging has several advantages over other modalities in evaluating osteoporosis patients, as it offers high-resolution 3D bone microarchitecture [19] that could then be analyzed quantitatively. The latter could then be used to compare therapeutic effects of proposed treatments. In this protocol, the vertebral defects were monitored via a sequence of in-vivo µCT scans at set time points for longitudinal assessment of bone repair. The vertebrae were then contoured and separated from the rest of the scan. A commercially available multiple image registration algorithm facilitated the extraction of an anatomically corresponding baseline VOIs to all subsequent time points. This technique provided a highly accurate and straightforward longitudinal 3D µCT analysis that is not user-dependent.
The methods described here for longitudinal assessment of bone repair using in-vivo µCT scans and a registration algorithm could be applied to any bone defect regeneration analysis. The vertebral defect model used here is a convenient model for this application, as its bone structure is unique and can be easily registered to the same anatomical position. However, any bone regeneration could be analyzed under the same conditions by properly separating the same bone of interest throughout the longitudinal scans. It is imperative to have the separated bone samples with the same anatomical features included. The anatomic match obtained by the registration procedure can only occur if the samples include the same anatomical features. The registration will allow the user to apply the exact predefined VOI of the first scan to all remaining time points, resulting in a highly accurate 3D histomorphometric analysis over time. Bone volume density and apparent density of the VOI can be used to assess new bone formation.
The experimental design of this protocol is shown in figure 1. Briefly, six week-old rats with their ovaries surgically removed were purchased and subjected to a special LCD consisting of 0.01% calcium and 0.77% phosphate. After a period of 4 months of LCD, the rats were operated on and a critical-size vertebral defect was drilled in the fourth and fifth lumbar vertebral bodies (L4–L5). Following surgery, the rats were imaged on day 1, week 2, 4, 8 and 12 after defect establishment. Defect margins of day 1 scans were located and reoriented to a standard position and a cylindrical VOI was defined. Subsequent µCT scans (i.e., for weeks 2, 4, 8 and 12) of each rat were automatically registered to standard position defined for the corresponding day 1 scan using commercially available software. Day 1 predefined VOI was applied to registered scans. Bone volume density and apparent density of the VOI were used to assess new bone formation.
• Ovariectomized athymic rats (Harlan Laboratories, Indianapolis, IN).
• Low calcium food (Newco Distributors, Inc., CA)
• Isoflurane (Patterson Veterinary)
• Phosphate Buffered Saline (PBS, Sigma-Aldrich, St. Louis, MO)
• Betadine (Generic)
• Chlorhexidine Gluconate 0.5% (Generic)
• Ethanol, 100% (Ethyl Alcohol 70% (vol/vol), Nedalco)
• Carprofen (Rimadyl, Zoetis, Florham Park, NJ)
• Ethicon mini Dermabond (Ethicon, Somerville, NJ)
• Buprenorphine hydrochloride (BD Pharmaceuticals)
• Anesthetics machine (Vet Tech Solutions)
• Ventilator (Active scavenger unit, Vet Tech Solutions)
• Induction chamber (Kent Scientific)
• Heating Pad (Rothacher Medical)
• Surgical drape (Medline Industries)
• Magnetic fixator retraction system (Fine Science Tools, Inc., CA)
• Microdissection scissors, 3.5 inch (Roboz Surgical Instruments)
• Scissors (Aesculap)
• Forceps, no. 5 (Roboz Surgical Instruments)
• Hemostatic forceps, straight, 3.5 inch (Roboz Surgical Instruments)
• Sterile cotton gauze (Klinion)
• Cotton swabs (sterile; EMRO Medical)
• Syringe, 1 ml (BD Plastipak)
• Needle, 25-gauge (BD Microlance)
• Laminar flow hood
• Thermal Cautery (World Precision Instruments, FL)
• Micro-Drill (Fine Science Tools, CA)
• Trephines for Micro Drill, 2mm diameter (Fine Science Tools, CA)
• 3-0 Vicryl undyed 27" SH taper (Ethicon, Somerville, NJ)
• 4-0 Ethilon black 18" PC-3 conventional cutting (Ethicon, Somerville, NJ)
• Cone-beam in vivo microCT (vivaCT 40, Scanco Medical was used in this protocol)
• Analyze software (Version 11.0; Biomedical Imaging, Mayo Clinic, Rochester, MN)
Induction of osteoporosis
1) Put six week-old athymic ovariectomized rats on a 4 months of low calcium diet (LCD) consisting of 0.01% calcium and 0.77% phosphate. Then, switch back to normal diet. These rats will be referred to as “osteoporotic rats” hereafter.
CAUTION: Research protocols involving the use of animals should be reviewed by the investigator’s institutional ethical review board to avoid any unnecessary discomfort or pain to the animals and to determine whether alternatives exist to animal research. All animal experiments should be performed in accordance with relevant guidelines and regulations of protocols approved by the investigator’s institutional animal research review committee. Personnel should be trained in animal handling.
Vertebral defect model
2) Anesthesia. Place the osteoporotic rat in an induction chamber of an anesthesia machine with a central scavenging system. Induce anesthesia using 5% isoflurane in 100% oxygen and maintain via nose cone at 2-3% isoflurane.
3) Apply toe pinch stimulus to ensure adequate plane of anesthesia. If no response is noted, the procedure can be initiated.
4) Preoperative care. Place the rat in dorsal recumbency on a heating pad (37 °C).
5) CRITICAL STEP: The temperature of the heating pad is important to prevent hypothermia since an anesthetized rat is unable to regulate its body temperature. Stretch the rat limbs using a magnetic fixator retraction system (Fig. 2A)
6) Shave the abdominal area and swab it with Betadine and Chlorhexidine Gluconate 0.5% followed by 70% ethanol.
7) Inject the rat with Carprofen (5 mg/kg BW, SQ) before beginning the surgical procedure.
8) Surgical procedure. Use sterile surgical scissors to cut the skin. Begin the incision 1cm below the xiphoid process and cut through the midline (~5-8 cm) (Fig. 2B).
CAUTION: Avoid using a scalpel for abdominal incisions. Using surgical scissors, as opposed to a scalpel, reduces bleeding and damage to underlying tissues.
CAUTION: All surgical tools must be autoclaved a day before surgery. In case of multiple surgeries, appropriate sterilization must be applied to all surgical tools. Between surgeries, wash the tools and place them in a sonicator bath for 5 minutes. Then, spray the surgical tools with Isopropyl Alcohol 70% and dry them. Next, place them for 20 seconds in a hot bead sterilizer set to 250°C. Allow the tools to cool down for 5 minutes. Now, the surgical tools can be used for the next surgery.
9) Use surgical scissors to make an incision of the aponeurosis through the linea alba to access the abdominal cavity. (Fig. 2C).
10) Use retractors to expose the abdominal cavity (Fig. 2D).
11) Deflect the intestines to the rat’s right to expose the abdominal aorta and the left kidney (Fig. 2E). Palpate the lumbar spine before proceeding to expose it.
CAUTION: To avoid dehydration, use sterile saline soaked gauzes to wrap the internal organs.
12) Use thermocautery to expose the anterior aspect of lumbar vertebral bodies and isolate them from surrounding connective tissue and muscles (Fig. 2F-G).
CAUTION: Use a cotton swab saturated with 3% hydrogen peroxide to remove blood and residual tissue from the vertebrae.
13) Use a sterile drill bit (~2mm in diameter) to drill a 5mm deep bone defect in the center of the vertebral body (Fig. 2H-I).
CRITICAL STEP: Apply minimal pressure to drill through only the ventral cortex and underlying trabecular bone and avoid drilling through the dorsal cortex. Note that the vertebrae of osteoporotic rats are very fragile.
CAUTION: Use a cotton swab to clean the defect and apply pressure to stop bleeding if present.
14) Repeat step 13 on an adjacent vertebra, to create a total of 2 defects per rat (Fig. 2J).
15) Return the intestines to the abdominal cavity.
16) Use a vicryl synthetic absorbable surgical suture (3-0 Vicryl undyed 27" SH taper) in a continuous pattern to suture the aponeurosis (Fig. 2K).
17) Close the skin using a monofilament nylon non-absorbable suture (4-0 Ethilon black 18" PC-3 conventional cutting) in a simple interrupted pattern (Fig. 2L).
18) Apply 100ul of Dermabond on top of the skin sutures and between them to ensure complete closure of the skin.
19) Postoperative care. Inject the rat with warm (37°C) lactated ringer’s solution (1CC/100g BW, SQ) to prevent hypothermia and dehydration.
20) Inject the rat with Buprenorphine (0.5 mg/kg, SQ) for post-op pain relief.
21) After the animal is recovered on the heating pad, return it to its cage.
CAUTION: House the rats individually, in separate cages, to prevent rat-to-rat mutilation of the sutures and wound.
22) Place chow soaked in water in a petri dish on the cage floor for a few days post-op to help the rats reach the food.
23) Administer Carprofen (5 mg/kg BW, SQ) 24 hours post-surgery for pain relief.
24) Remove skin sutures while animal is under 2% isoflurane anesthesia 10-14 days post-op.
MicroCT scanning
25) One day after the surgical procedure, place the osteoporotic rat in an induction chamber of an anesthesia machine with central scavenging system. Induce anesthesia using 5% isoflurane in 100% oxygen and maintain via nose cone at 2-3% isoflurane.
26) Scan the rat using an in-vivo microCT scanner. Repeat scanning for longitudinal analysis of bone regeneration.
CRITICAL STEP: Make sure that all animals are scanned using the same settings (X-ray energy, scanning medium, intensity, voxel size and image resolution) and in a similar orientation. Refer to Bouxtein et al[1] for further explanations and considerations involved in rodent microCT scanning for assessment of bone microstructure.
Vertebral separation
27) Contour the vertebra of interest, as demonstrated in Fig. 3A-I. Make sure to include all parts of the vertebra while excluding parts that belong to adjacent vertebrae.
28) Save the contoured vertebra as a separate file (Fig. 3J-K).
Definition of the VOI for longitudinal quantitative evaluation
29) The following steps depend on whether the scan is from day 1 after surgery (reference vertebra) or from the subsequent time points (target vertebrae).
(A) Reference vertebra
\(i) Z rotation. Measure the angle of the margins using a xy slice from the center of the defect \(Fig. 4A-B). Use the measured angle to rotate the vertebra \(Fig. 4C).
\(ii) X rotation. Measure the angle of the margins using a yz slice from the center of the defect \(Fig. 4D-E). Use the measured angle to rotate the vertebra \(Fig. 4F).
\(iii) Flip. Flip the rotated vertebra by changing the xy plane to the zx plane.
\(iv) VOI definition. Draw a circular contour of the defect using a slice from the center of the defect \(Fig. 6A). Then, copy that contour and paste it on all the slices in the defect \(Fig. 6B). CRITICAL STEP: Since all defects were created using the same procedure, the same number of slices and subsequently total volume (TV) should be analyzed for all samples.
(B) Target vertebra
\(i) Load the DICOM files of both the target and the reference vertebrae to the main window of the “Analyze” software. CRITICAL STEP: To avoid gray scale value changes, define the same output data type as the original DICOM files in the load menu.
\(ii) Registration to reference vertebra. Launch the “3-D Voxel Registration” module, and input the reference vertebra as the “Base Volume” and the target vertebra as the “Match Volume”. Click “Register” to register the vertebrae \(Fig. 6).
\(iii) Save the registered file using the same data type and import it to your microCT environment.
\(iv) Application of VOI. Apply the VOI defined for the reference vertebra to the registered target vertebra. Check that the VOI and defect are concentric. MicroCT analysis
30) Send the VOI for evaluation using your microCT evaluation program.
CRITICAL STEP: Make sure to use the same parameters when analyzing all VOIs. Make sure the threshold is set high enough to omit background noise with minimal loss of bone.
Steps 2-24, Vertebral defect model: 40-50 minutes
Steps 25-26, MicroCT scan: 30-40 minutes
Steps 27-28, Vertebral separation: 20-30 minutes per sample
Step 29, VOI definition: 20-30 minutes per reference vertebra, 10-20 minutes per target vertebra
Step 30, MicroCT analysis: 10-20 minutes
See Figures section for a table giving troubleshooting guidance.
Using this protocol, one can image and quantify the regeneration of modeled osteoporotic vertebral defects across different time points. The anatomic match obtained by the registration procedure allows the analysis of the exact same VOI at all time points. Resulting in a highly accurate longitudinal 3D histomorphometric analysis even when the margins of the original defect are no longer recognizable. We used five time points (Day 1, Week 2, Week 4, Week 8 and week 12) as an example for longitudinal evaluation of bone regeneration (Fig. 7). Regenerated can be evaluated by both qualitative assessment of 2D cross-sections and 3D images (as illustrated in Figure 7A), and quantitative comparison of the bone quantity (BVD) and quality (AD) (Fig. 7B). The following morphometric indices can be determined for newly formed bone in regeneration sites: (i) TV of bone, including new bone and soft-tissue cavities (TV, mm3); (ii) volume of mineralized bone tissue (BV, mm3); (iii) bone volume density, which can also be referred to as the percentage of regeneration for the round critical-size bone defect model (BV/TV); and (iv) bone mineral density (BMD, mg hydroxyapatite per cm3), derived from comparison of X-ray attenuation in the scanned bone sample with that of hydroxyapatite standards.
- Wang, M. L. et al. A rat osteoporotic spine model for the evaluation of bioresorbable bone cements. The spine journal : official journal of the North American Spine Society 7, 466-474, doi:10.1016/j.spinee.2006.06.400 (2007).
- Consensus development conference: prophylaxis and treatment of osteoporosis. The American journal of medicine 90, 107-110 (1991).
- Sheyn, D. et al. PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries. Mol Ther, doi:10.1038/mt.2015.211 (2015).
- Center, J. R. et al. Mortality after all major types of osteoporotic fracture in men and women: an observational study. Lancet 353, 878-882, doi:10.1016/S0140-6736(98)09075-8 (1999).
- Buchbinder, R. et al. A randomized trial of vertebroplasty for painful osteoporotic vertebral fractures. The New England journal of medicine 361, 557-568, doi:10.1056/NEJMoa0900429 (2009).
- Kallmes, D. F. et al. A randomized trial of vertebroplasty for osteoporotic spinal fractures. The New England journal of medicine 361, 569-579, doi:10.1056/NEJMoa0900563 (2009).
- Kado, D. M. et al. Vertebral fractures and mortality in older women: a prospective study. Study of Osteoporotic Fractures Research Group. Arch Intern Med 159, 1215-1220 (1999).
- Silverman, S. L. The clinical consequences of vertebral compression fracture. Bone 13 Suppl 2, S27-31 (1992).
- Ross, P. D. Clinical consequences of vertebral fractures. Am J Med 103, 30S-42S; discussion 42S-43S (1997).
- Vanecek, V. et al. The combination of mesenchymal stem cells and a bone scaffold in the treatment of vertebral body defects. European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society 22, 2777-2786, doi:10.1007/s00586-013-2991-2 (2013).
- Liang, H. et al. Use of a bioactive scaffold for the repair of bone defects in a novel reproducible vertebral body defect model. Bone 47, 197-204, doi:10.1016/j.bone.2010.05.023 (2010).
- Kobayashi, H. et al. Long-term evaluation of a calcium phosphate bone cement with carboxymethyl cellulose in a vertebral defect model. J Biomed Mater Res A 88, 880-888, doi:10.1002/jbm.a.31933 [doi] (2009).
- Fujishiro, T. et al. Histological evaluation of an impacted bone graft substitute composed of a combination of mineralized and demineralized allograft in a sheep vertebral bone defect. J Biomed Mater Res A 82, 538-544, doi:10.1002/jbm.a.31056 [doi] (2007).
- Sheyn, D. et al. Gene-modified adult stem cells regenerate vertebral bone defect in a rat model. Molecular pharmaceutics 8, 1592-1601, doi:10.1021/mp200226c (2011).
- Wang, M. L. et al. Altered bioreactivity and limited osteoconductivity of calcium sulfate-based bone cements in the osteoporotic rat spine. The spine journal : official journal of the North American Spine Society 8, 340-350, doi:10.1016/j.spinee.2007.06.003 (2008).
- Liang, H. et al. A novel strategy of spine defect repair with a degradable bioactive scaffold preloaded with adipose-derived stromal cells. Spine J 14, 445-454, doi:10.1016/j.spinee.2013.09.045 (2014).
- Phillips, F. M. et al. In vivo BMP-7 (OP-1) enhancement of osteoporotic vertebral bodies in an ovine model. The spine journal : official journal of the North American Spine Society 6, 500-506, doi:10.1016/j.spinee.2006.01.014 (2006).
- Geusens, P. et al. High-resolution in vivo imaging of bone and joints: a window to microarchitecture. Nature reviews. Rheumatology 10, 304-313, doi:10.1038/nrrheum.2014.23 (2014).
- Genant, H. K., Engelke, K. & Prevrhal, S. Advanced CT bone imaging in osteoporosis. Rheumatology 47 Suppl 4, iv9-16, doi:10.1093/rheumatology/ken180 (2008).
Associated Publications
PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries
by Dmitriy Sheyn, Galina Shapiro, Wafa Tawackoli, +16
Molecular Therapy (16 March, 2016)