In Vitro Study: Experimental Design
To evaluate the effect of injection temperature on the final rheologic properties of the collagen-platelet gels, composites were manufactured under specified experimental conditions using a specifically designed manufacturing device () and then injected directly onto the plate of a small oscillation rheometer. Gelation was allowed to progress to completion as defined by a plateau in the elastic modulus values. Changes in elastic and inelastic modulus and time to the visco-elastic transition and the plateau in elastic modulus were recorded for all samples ().
Figure 1 Device for controlled mixing and heating of the collagen composites. The components of the composite were drawn up the syringe using the collapsible auger. The syringe was then placed in the cradle and attached to the motor via a hex connection on the (more ...)
Gelation result for a collagen-platelet composite mixed for 30 s at 100 rpm, and heated at 0.05°/s.
Manufacturing of Acid-Soluble Collagen Used in Composites
The collagen used in this study was derived from rat tails that were obtained from control breeder rats undergoing euthanasia for other Institutional Animal Care and Use Committee approved studies at our institution. The rat-tail tendons were sterilely harvested, minced, and solubilized in 0.01 N hydrochloric acid. The collagen content in the resulting solution was found to be >5 mg/mL. Previous collagen slurries obtained using the same in-house methodology were shown to be made mostly of type I collagen based on their amino acid profile and SDS-PAGE migration pattern. The same collagen solution was used in all experiments.
Prior to testing, the collagen solution was combined with HEPES Buffer (Cellgro; Mediatech, Inc., Herndon, VA), Ham's F-10 medium (MP Biomedicals, LCC, Aurora, OH), antibiotic-antimycotic solution (Cellgro), and sterile water. Sodium bicarbonate (7.5%; Cambrex BioScience Walkersville, Inc., Walkersville, MD) was used to neutralize the acidic mixture to a pH of 7.4. The neutralized collagen solution was kept on ice until use.
Preparation of Platelet Solution
Five hundred milliliters of whole blood was drawn from each of two hematologically normal pigs undergoing other Institutional Animal Care and Use Committee approved studies for the rheologic studies and autologously for each of the five animals in the in vivo studies. Blood was collected in a bag containing 10% by volume acid-citrate dextrose as an anticoagulant and transferred to centrifuge tubes. The blood samples were centrifuged for 6 min at 150 ×g (GH 3.8 rotor, Beckman GS-6 Centrifuge Beckman Coulter, Inc., Fullerton, CA). The supernatant was collected as platelet concentrate. Complete blood counts (CBCs) were measured for the whole blood and platelet concentrate. In vivo, the platelet solution was stored at room temperature for less then 30 min prior to addition to the neutralized collagen.
Preparation of Gels
One milliliter aliquots of the acid soluble collagen were vortexed with buffer to neutralize the solution. This solution aspirated into a syringe containing a collapsible auguer. One milliliter of platelet concentrate was then aspirated into the same syringe. The syringe was affixed in the cradle and the auger engaged with the mixing motor and the gel heated and mixed according to the specified test conditions as detailed below. Mixing speed, mixing time, and heating rate were controlled using a device made for this testing. (; TNCO, Inc., Whitman, MA). An auger was designed to fit inside the 6 cc syringe held in the cradle. This allowed for mixing of the collagen composite components while simultaneously warming the composite. This device had a motor that was coupled to the auger to allow for control of mixing speed and time, and a heating pad under the syringe that allowed for control of heating rate. The device was driven by a custom LabView (Austin, TX) application that allowed for control of the variables, and logging of feedback data.
Composites were created using three different mixing speeds (50, 100, and 150 rpm), three different mixing times (30, 60, and 120 s), and three different heating rates (0.05, 0.10, and 0.15°/s). All combinations of those parameters were tested in triplicate (19 cases total). Control gels were also tested, mixed for 30 s at 100 rpm, without heating. The final temperature of the gels was recorded for all mixing conditions. Additional triplicate gels having an injection temperature of 24°C–26°C, 26°C–28°C, 28°C–30°C, and 30°C–32°C were also tested. The additional gels were prepared by mixing at 100 rpm and heating at 0.10°/s for the time necessary for the gel to reach the specified final temperature.
In Vitro Rheology
Rheological properties of the gels were determined using Cone on Plate Small Amplitude Oscillatory Shear Rheometry using a TA Instruments AR 1000 Rheometer (New Castle, DE). The rheometer was fitted with a 60 mm 1° acrylic cone, and the base plate was maintained at 25°C. For each test, 1 mL of the CPCs was dispensed onto the rheometer plate. The cone was lowered so that the composite was situated in a 38 μm layer between the cone and plate, subjected to a 1% oscillatory. Strain (γo) with an angular frequency of 6.3 rad/s, and the resultant stress (τ(τ)) were recorded. The stress waveform is broken down to one waveform in phase with the oscillatory strain (τ′), and one waveform 90° out of phase (τ″), with the relationship being: τ=τ′+τ′=τo'sinωt+τo″cosωt. The visco-elastic complex modulus (G*) of the gel can be derived from the relationship G*(t) = G′(t) + iG″(t), where G′(t) = τo′/γo and G″(t) = τo″/γo, with the elastic modulus representing the elastic portion of the scaffold, and the inelastic modulus representing the viscous component. Phase angle (δ), which represents the lag between the applied strain and the resultant stress, is determined by the geometric relationship tan(δ) = G″/G′. A value of 45° represents the intersection of G′(t) and G″(t) and is defined as the visco-elastic transition point. Data points for elastic modulus, inelastic modulus, and phase angle were collected at 0.1 Hz, until the rate of change of the increase in elastic modulus was less then 0.1% for three consecutive data points, which was the mathematical definition of the plateau. A sample result is shown in .
In Vivo Studies: Experimental Design
Five 30 kg female Yorkshire pigs were used to study the effect of injection temperature on the in vivo performance of the CPCs. Four animals had bilateral ACL transections and for each of these, one side was treated with a suture repair augmented with a collagen sponge containing a CPC injected at a temperature ranging from 28.9°C–32.4°C, while on the contralateral side, the transection was treated with suture repair with the collagen sponge carrier only. A CPC was used because prior in vivo and in vitro studies have shown gradual platelet activation and platelet growth factor release from these CPCs.20,21
In the remaining animal, unilateral surgery was performed with the augmented repair and the contralateral side left as a contemporary intact control. One of the animals developed a postoperative seroma. The seroma was treated with prophylactic antibiotics until complete wound closure was observed on the collagen-platelet side. This knee was excluded from the study. Therefore, there were a total of four knees in the augmented repair group and four knees in the nonaugmented group. All animals were survived to 14 weeks and then underwent MRI evaluation and euthanasia. Knees were immediately harvested and frozen until biomechanical testing. Load to yield, load to failure, maximum linear stiffness, and displacement to failure were measured.
Institutional Animal Care and Use Committee approvals were obtained prior to beginning the study. Five 30 kg female Yorkshire pigs were used anesthetized per an approved protocol. After anesthesia had been obtained, the pigs were weighed and placed in the supine position on the operating room table. Both hind limbs were shaved, prepared with chlorhexidine followed by betadyne paint, and sterilely draped. No tourniquet was used. To expose the ACL, a 4 cm incision was made over the medial border of the patellar tendon. The incision was carried down sharply through the synovium using electrocautery. The fat pad was released from its proximal attachment and partially resected to expose the intermeniscal ligament. The intermeniscal ligament was released to expose the tibial insertion of the ACL. The anterior horns of the medial and lateral menisci attach separately from the intermeniscal ligament and therefore severing this ligament did not lead to gross meniscal instability. A Lachman maneuver was performed prior to releasing the ACL to verify knee stability. Two #1 Vicryl sutures were secured in the distal ACL stump at the middle and distal thirds of the ACL using a modified Kessler stitch. The ACL was transected completely at the junction of the middle and proximal thirds using a #12 blade. No medial collateral ligament (MCL) transection was performed. Complete transection was verified visually and a repeat Lachman maneuver was positive in all knees with no significant endpoint detected after complete transaction (knee was unstable). An absorbable suture anchor (TwinFix AB 5.0 Suture Anchor with DuraBraid Suture [USP#2]; Smith and Nephew, Inc., Andover, MA) was placed at the back of the femoral notch. The knee was irrigated with 500 cc of sterile normal saline to remove all synovial fluid. Once hemostasis had been achieved, a 1 cm ×1 cm collagen sponge was threaded onto the sutures of the suture anchor and slid up into the intercondylar notch. In prior studies, the use of the collagen sponge combined with platelet-rich plasma showed enhanced healing using this technique at 4 weeks.1
The DuraBraid sutures were then tied to the Vicryl sutures previously placed in the ACL stump using maximum manual tension with the knees in resting flexion (approximately 70°, which is 40° short of full extension in these animals). A batch of CPC was mixed by sequentially drawing up equal aliquots of neutralized collagen solution and autologous platelet concentrate into the mixing and heating device and mixing for 1 min at 50 rpm and 0.10°/s, which resulted in injection temperatures between 28.9°C and 32.4°C. The collagen-platelet mixture was then injected to fill the notch and area around the ACL repair (). The knee was left in resting extension and allowed to gel while the identical technique of suture anchor repair was performed with an identical collagen sponge, but without the addition of the CPC. Gelation was observed to have occurred within 10 min of gel injection by manual examination of the joint and by opacification of the composite. In the knees treated with suture repair and collagen aponge only, blood clot was seen to soak the collagen sponge in situ after suture anchor placement. The incisions were closed in multiple layers with absorbable sutures.
Schematic diagram of the suture anchor and repair to the ligament stump, with the collagen sponge in the gap and the collagen-platelet slurry injected around the repair construct.
The animals were not restrained postoperatively, and were allowed ad lib activity. Once the animals recovered from anesthesia, they were permitted to resume normal cage activity and nutrition ad lib. Buprenex 0.01 mg/kg IM once and a Fentanyl patch 1–4 mcg/kg transdermal were provided for postoperative analgesia. All animals were weight bearing on their hind limbs by 24 h after surgery. After 14 weeks in vivo, the animals were again anesthetized and underwent in vivo MR imaging using the protocol detailed below.
After the MR images had been obtained, the animals were euthanized using Fatal Plus at 1 cc/10 lbs. No animals had any surgical complications of difficulty walking normally, redness, warmth, and fever, or other signs of infection that would have necessitated early euthanasia.
Six intact control knees were obtained from age-, gender-, and weight-matched animals after euthanasia following surgical procedures to the chest. The hind limbs were frozen at −20°C for 3 months and thawed overnight at 4°C before mechanical testing. All other testing conditions for these knees were identical to those in the experimental groups.