1. Introduction
Pre-clinical studies for drug development are time consuming, expensive, and the number of animals’ lives lost in the process is astronomical. In general, animal models are not optimal models, because they are not synonymous with the anatomy and physiology of humans. There is a need for another model for drug development. The human skeletal system could be the optimal model because bones contain a plethora of stem and bone cells. The skeleton also contains bone marrow, which is sensitive to toxicity. Bone tissue is very structurally sound and rigid, therefore, there is no need for additional external support that would be required for other organs. Testing drugs on a human femoral head ex vivo, would be ideal because the direct effects could be observed, and the overall efficacy and outcome of future clinical trials could be improved [
1,
2]
Alternative drug testing systems use biomaterials and synthetic scaffolds that are utilized within a perfusion chamber. A dynamic laminar fluid flow system was achieved through the use of a peristaltic pump, tubing, and a medium reservoir. This allows for transport of key nutrients and oxygen throughout the system [
3]. However, the scaffolds and biomaterials used to seed the cells, do not contain any vasculature and contain only the trabecular part of the bone. Most of the time only a limited number of cell types are utilized for these studies [
4,
5,
6,
7].
In this study, we perfused human femoral heads using a self-designed perfusion bioreactor that sustained cell viability in an ex vivo human femoral head. By cannulating the four main arteries found in the human femoral head, the medial and lateral circumflex arteries and the posterior superior branch, and the posterior inferior branch of the retinacula arteries, we were able to supply the entire femoral head with nutrient-filled media. The media flowing through the bone would provide the bone with the required flow rates, mass transfer, nutrients, pH, and gases. These elements, along with other factors, like temperature, humidity, and removal of toxic waste, will allow for maintaining the femoral head in the dynamic environment required to survive ex vivo.
2. Materials and Methods
2.1. Bioreactor Design
The perfusion bioreactor was devised to be a closed system. The peristaltic pump P1 (General Electric, Piscataway, NJ, USA) was used to conserve the constant flow of media throughout the bioreactor. A total of 1.75 L of Dulbecco’s Modified Eagle’s Medium (Corning, New York, NY, USA), (with 10% (
v/
v) fetal bovine serum (FBS) (Gemini Bio-Products, West Sacramento, CA, USA), 1% (
v/
v) penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, USA), and 1% antibiotic-antimycotic (Gemini Bio-Products, West Sacramento, CA, USA)), was used to perfuse the system for 12 h.
Figure 1 illustrates the flow system of the bioreactor. A peristaltic pump was used to create a perfusion rate of 6–8 mL/min. The pump was connected to the manifold with silicone tubing (ID 1/4" × 3/8” wall thickness) and a final image of the Bioreactor system can be seen in
Figure 2A. The manifold branched into four tubes (3/16” ID), which were connected to 21 ½ gauge hypodermic needles that cannulate the femoral head arteries and allow media to perfuse the bone sample (
Figure 2C,D). A pressure gauge and relief valve were added to the manifold to maintain a constant pressure and to avoid pressure build-up or backflow (
Figure 2B). A pressure range of 0.1–1.1 psi was maintained to mimic the flow pressures in bone. To allow a continuous and sterile exchange of gases, a Breathe Easy® gas permeable sterile film was used (USA Scientific, Ocala, FL, USA). The direction of flow is as follows: from the peristaltic pump, to the manifold, to the femoral head vasculature, from the femoral head vasculature into the media chamber, then back into the pump.
2.2. Pre-Soaking Assay to Reduce Pressure Build-Up within the Femoral Heads
Bovine femoral heads were collected from Herman’s Meat Shop in Newark, DE, USA and were incubated in varying concentrations of mannitol (5% mannitol (w/v) for one hour, 10% mannitol (w/v) for one hour, 15% mannitol (w/v) for one hour, and 10% mannitol (w/v) for two hours) or (0.15% heparin for one hour, 0.0025% of heparin for one hour, and 0.15% of heparin for two hours). All solutions were created in DMEM (Corning, New York, NY, USA), supplemented with 10% (v/v fetal bovine serum (FBS) (Gemini Bio-Products, West Sacramento, CA, USA), 1% (v/v) penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, USA), and 1% Antibiotic-Antimycotic (Gemini Bio-products, West Sacramento, CA, USA).
2.3. Villanueva Stain to Show Viable Perfusion
It was important to determine whether DMEM was flowing through the vasculature of the bovine femoral head. Therefore, a 1% (v/v) solution of Villanueva stain was prepared using 1X PBS, which was run through the bioreactor for a total of 12 h. After 12 h, the samples were sliced into 2–2.5 mm slices using a modified tile saw and the stain was compared to an image of a control bone (that was incubated in the same solution, but not perfused). The Villanueva stain determined the viable perfusion in the femoral head, through the vasculature.
2.4. Cell Viability Assay with Mannitol and Heparin
Cell viability was also tested using CellTrace™ Calcein red-orange AM. A Hoechst nuclear stain was also used in order to determine total number of cells, as it stains the nucleus of both live and dead cells. C2C12 cells were used and treated with varying concentrations of both mannitol (5%, 10%, and 15%) and heparin (0.15% and 0.0025%) for one or two hours. C2C12 cells in DMEM supplemented with 10% FBS, and 1% penicillin/streptomycin at 37 °C in an environment of 5% CO2/95% air were grown. Once ready for treatment, the cells were seeded at 1.0 × 105 cells/mL in a 24 well plate for 12 h before treatment. The growth media was then replaced and supplemented with 1 µM of Calcein red-orange AM in addition to the designated treatment. After the corresponding time point (either one or two hours post treatment) the cells were stained with Hoechst for 2.5 min at room temperature and immediately counted for cell viability. Percent viability was then calculated. This was a standard 2D culture experiment.
2.5. Femoral Head Cannulation and Perfusion
Human femoral heads were obtained from female patients with osteoporosis who had undergone total hip replacement surgery at Christiana Care Hospital in Newark, DE, USA. The specimens were kept at 4 °C until use. The specimens were incubated in 10% (w/v) mannitol in DMEM. Then, a surgical scalpel was used to cut back the neck of the femoral head, which uncovered the four main arteries (or cannulation sites). If cannulation was obstructed, a 21 gauge, 1-1/2-inch needle drill bit was used to hold the needle in its place. To avoid introducing air bubbles into the bone, the tubing was not attached to the cannulation sites until the media was thoroughly pushed through the tubing. The bioreactor was then run for 12 h. After 12 h, the bioreactor was stopped and the samples were processed. The control specimen was an osteoporotic femoral head that was not perfused, but was kept in the same DMEM medium for 12 h in order to determine diffusion of DMEM into the sample.
2.6. Parameters Affecting the Uptake of Nutrients and Media Flow
Changes in flow, pressure, glucose levels, and pH of the media were determined because these parameters are important in regards to successful perfusion. If the pressure increased or decreased, this would mean that media was coagulating within the bone. If the glucose levels were to decrease this would signify that the cells within the bone were still alive and taking up vital nutrients. pH was checked to verify that no contamination had occurred, as increases in pH have been linked to contamination [
8]. A pressure gauge (Dwyer Instruments, Michigan City, IN, USA) was used and readings were taken every hour in triplicate, pH was measured hourly and was approximated using pH paper. Glucose was measured every three hours using a One Touch Glucose Monitor (LifeScan, Milpitas, CA, USA).
2.7. Obtaining Human Femoral Head Bone Slices
Once the bioreactor was stopped and the human femoral head was removed, cores (using a 3/8” drill bit core, cores were 1.5–2 cm in length) of the specimen were taken down the midsagittal plane and immediately placed in DMEM. The femoral head was contained in 1X PBS in order to avoid overheating while the core was taken. These cores were then sectioned using a miter saw (also in 1X PBS). Three slices (about 500 microns) were obtained from the top, middle, and bottom of the core and labeled as such. This was to determine the stain penetration in comparison to perfusion versus diffusion.
2.8. Image Analysis of Ex Vixo Cell Viability in Human Femoral Heads
Images of the human bone slices were taken using LSM 880 Zeiss Microscope (Zeiss, Oberkochen, Germany) with an LD LCI plan-apochromat 25×/0.8 l mm korr DIC M27 objective (Zeiss, Oberkochen, Germany). Two lasers (561 nm at 10–22% power and 405 nm at 1.1–1.8% power) were used and images were taken as a Z-stack. A spectral scan was run and collected on a control bone that was stained with 1 µM of Calcein red-orange AM (Calcein acetoxymethyl ester). This was to establish the spectral peak of the dye (590 nm emission spectrum) and was then set as the control peak. Linear mixing was used, in order to identify the defined spectrum and remove autofluorescence of the stain (in some cases fingerprinting technology was also used).
2.9. Statistical Analysis
When comparing two groups, a Student’s t-test was used with p < 0.05 denoting significance. Comparisons of more than two datasets were analyzed using a one-way ANOVA with a Tukey Kramer post-hoc test. Statistical significance was shown if p < 0.05.
4. Discussion
Bioreactors for tissue engineering are still in the early stages of development. A tissue engineering bioreactor must: (1) have even cell distribution throughout the scaffold; (2) satisfy physiologically-relevant factors (like nutrients, oxygen, pH, removal of waste, temperature, and humidity) that is needed by the tissue [
17]; (3) aid in the mass transport of medium throughout the scaffold; and (4) integrate required physical parameters (like the flow rate of the blood within bone, pressure, fluid shear stress, resistance, and compliance). The bioreactor should also be effortlessly monitored, controlled, and sterile, in addition to the results being reproducible [
1]. Our perfusion bioreactor was designed to address these parameters. Silicone tubing was used throughout the system because it is a commonly used biomaterial in implants, biocompatible, and can be autoclaved at 121 °C for 15 min [
18]. A manifold was designed using stainless steel because previous studies have shown that long-term use does not increase toxicity [
17]. Within the manifold there were a pressure gauge and pressure relief valve. These were used to adjust the pressure levels if it was outside the normal, physiological range of 0.1–1.1 psi [
19,
20]. Syringe needles (21 ½ gauge) were used to cannulate the bone, because their width (0.5 mm) matched the inner diameter of four main arteries (0.5–8.5 mm).
The perfusion flow rate within the femoral heads were maintained at 6–8 mL/min. This is comparable to the in vivo blood flow rate in the bone, which is approximately 5–20 mL/min [
21]. We ensured bone viability through perfusion of media into the pre-existing vasculature found within the femoral head. Calcein red-orange AM is a red orange dye that is intrinsically fluorescent and can permeate through the plasma membrane of live cells, in part, due to its a small molecular weight (789.55 g/mol). A concentration of 100 nM was mixed into the media that was perfused through the bone.
In order to extend the lifespan of the femoral head ex vivo, several trouble-shooting experiments were conducted in order to optimize the efficacy of our bioreactor. Pressure-relieving agents (mannitol and heparin) were tested in order to ensure continuous media flow through the femoral head and the results are shown in
Figure 3A,B. Heparin inhibits the activity of thrombin, which is accountable for removing blood clots. However, heparin also activates the enzyme lipoprotein lipase in the blood. Heparin-soaked bones were noticeably softer than the mannitol-soaked bones. The previously described enzymatic activity is a possible explanation for the softening of the bone [
11]. Mannitol increases the osmolarity of the medium and effectively removed clots formed, aiding in keeping the flow of media constant, without altering the integrity of the bone [
22]. Continued, long-term use of mannitol has been shown to increase hypertension, therefore, shorter time points were selected [
16]. The effect of mannitol and heparin on cell viability demonstrated that mannitol was the more effective supplement (
Figure 3C,D). In addition, a Villanueva stain was used in order to show the successful and viable perfusion through a bovine femoral head when compared to a non-perfused bovine femoral head. This stain showed that the perfusion had occurred successfully through the vasculature, and that apoptosis due to re-perfusion injury was minimal.
The human femoral heads used were obtained from female osteoporotic patients between the ages of 80–90 and were collected from Christiana Care Hospital within 24 h of hip arthroplasty surgery. Previous in vitro experiments using human specimens have shown the presence of some live cells within 24 h of procurement [
23], therefore, it would be possible to obtain live cell images from a perfused femoral head after 12 h of perfusion. The presence of live cells and the perfusion of bone are shown in
Figure 5A. The Calcein red-orange stain was more apparent throughout the perfused bone, which was not observed in the control. The pressure was maintained between 0.1–1.1 psi, while the sugar levels increased, and then subsequently decreased, over time. The increase in sugar could be attributed to the mannitol being released from the femoral head, and the subsequent decrease could suggest that it was being utilized by the cells of the femoral head (
Figure 4A,B). The glucose meter utilized could detect multiple types of sugar (including mannitol), which could explain why there was an increase.
Limitations of this study include the number of trials conducted (three), the tissue (femoral head) used, and the time period the femoral head was perfused. In the future more trials across different bone tissue types should be investigated in order to obtain reliable data regarding the use of a perfusion bioreactor to study bone in vivo. The method in measuring sugar concentrations could also be improved upon, since a commercial glucose monitor was used, and could be attributed to the unexplained sugar spike at six hours. In addition, the bioreactor should be run for a longer period of time (24 h or 48 h) to confirm its effectiveness in maintaining cell viability ex vivo. Femoral heads from female patients aged 80–90 years old were used and, in the future, more trials should include femoral heads from both male and female patients, as well as patients at varying ages, in order to eliminate possible confounding variables.
In conclusion, this study shows that human femoral heads can be used as a biological platform and bone cell viability can be maintained ex vivo. The successful perfusion in human femoral heads was shown through the viability of cells 12 h post perfusion (
Figure 5A,B). This is a potential drug-testing model that could be invaluable in the future for osteoporotic therapeutic testing.