Selected Research Abstracts from Publications

Mesenchymal stem cell seeded, biomimetic 3D printed scaffolds induce complete bridging of femoral critical sized defects
Szivek JA, Gonzales DA, Wojtanowski AM, Martinez MA, Smith JL
J Biomed Mater Res B Appl Biomater, PMID: 29569331, DOI: 10.1002/jbm.b.34115, March 2018

Abstract: No current clinical treatments provide an ideal longtermsolution for repair of long bone segment defects. Incomplete healing prevents patients from returning to preinjury activity and ultimately requires additional surgery to induce healing. Obtaining autologous graft material is costly, incurs morbidity, requires surgical time, and quality material is finite. In this pilot study, 3D printed biomimetic scaffolds were used to facilitate rapid bone bridging in critical sized defects in a sheep model. An inverse trabecular pattern based on micro-CT scans of sheep trabecular bone was printed in polybutylene terephthalate. Scaffolds were coated withmicron-sized tricalcium phosphate particles to induce osteoconductivity.Mesenchymal stem cells (MSCs) were isolated from sheep inguinal and tail fat, in one group of sheep and scaffolds were infiltrated with MSCs in a bioreactor. Controls did not undergo surgery for cell extraction. Scaffolds were implanted into two experimental and two control adult sheep, and followed for either 3 or 6 months. Monthly radiographs and post explant micro-CT scanning demonstrated bone formation on the lateral, anterior, medial, and posterior-medial aspects along the entire length of the defect. Bone formation was absent on the posteriorlateral aspect where a muscle is generally attached to the bone. The 3-month time point showed 15.5% more cortical bone deposition around the scaffold circumference while the 6-month time point showed 40.9% more bone deposition within scaffold pores. Control sheep failed to unite. Serum collagen type-1C-terminus telopeptides (CTX-1) showed time-dependent levels of bone resorption, and calcein labeling demonstrated an increase in bone formation rate in treated animals compared with controls.

A Purification Technique for Adipose-Derived Stromal Cell Cultures Leads to a More Regenerative Cell Population
Conway RF, Okarski KM, Szivek JA
J Invest Surg, pp. 1-12, PMID: 29388858, ISSN: 0894-1939 print / 1521-0553 online, DOI: 10.1080/08941939.2017.1423420, Feb 2018

Abstract: It has been demonstrated that adipose-derived stromal cells (ASCs) are a regenerative cell population with potential uses for bone and cartilage regeneration. However, the biomarker expression and heterogeneity of the population has not been thoroughly characterized. By analyzing biomarker expression, we aimed to understand the composition of ASC populations extracted using a common extraction technique in comparison to ASC populations given an additional purification step. Human adipose tissue samples were collected, and ASCs were extracted from these samples using a common, published extraction technique (primary extraction). These cells were cultured and half were given an additional purification. The primarily-extracted and purified cell populations were analyzed for biomarkers that correspond to specific cell types. The addition of the purification technique reduced the number of cells expressing hematopoietic and endothelial biomarkers and did not cause the yield of mesenchymal stem cell biomarker-expressing cells to decrease. Biomarkers corresponding to erythrocytes and lymphocytes were lost during the primary extraction, and biomarkers corresponding to most granulocytes and progenitor cells were lost during the additional purification. Biomarkers identifying dendritic cells, monocytes/ macrophages, neutrophils, vascular endothelial cells, smooth muscle cells, and pericyteswere upregulated in purified cell populationswhile those identifying fibroblasts and adipocytes were downregulated. Pluripotency biomarkers were more highly expressed in purified cell populations. These results demonstrate that the most commonly utilized adipose tissue recovery and ASC extraction technique leads to a heterogeneous cell population in which further purification of this population, as described in this manuscript, isolates a cell subset that has more regenerative potential.

Determination of joint loads using new sensate scaffolds for regenerating large cartilage defects in the knee
Szivek JA, Ruth JT, Heden GJ, Martinez MA, Diggins NH, Wenger KH
J Biomed Mater Res B Appl Biomater, vol. 105, issue 6, pp. 1409-1421, PMID: 27080933 Aug 2017

Two complete unicondylar surface replacement scaffold designs to support tissue-engineered cartilage growth that utilized adult endogenous stem cells were 3D printed and tested in a dog stifle model. Integrated rosette strain gauges were calibrated and used to determine shear loading within stifle joints for up to 12 months. An activity index that compared extent of daily activity with tissue formation showed differences in the extent and quality of new tissue with the most active animal having the most new tissue formation. Shear loads were highest early and decreased with time indicating that cartilage tissue formation begins while tissues experience high shear loads and continues as the loads decrease toward normal physiological levels. Scaffolds with biomimetic support pegs facilitated the most rapid bone ingrowth and were noted to have more cartilageformation with better quality cartilage as measured using both indentation testing and histology. Comparison of implant placement depth to previous studies suggested that placement depth affects the amount of tissue formation. This study provides measurements of loading patterns and cartilage regeneration on a complete medial condylar surface replacement that can be used for preclinical testing of a tissue engineering approach for the most common form of early stage osteoarthritis, unicondylar disease.

In vivo telemetric determination of shear and axial loads on a regenerative cartilage scaffold following ligament disruption,
Szivek JA, Heden GJ, Geffre CP, Wenger KH, and Ruth JT,
J Biomed Mater Res B Appl Biomater, vol. 102, issue 7, pp. 1415-25, PMID: 24678004, Oct 2014

Abstract: Recent interest in repair of chondral and osteochondral cartilage defects to prevent osteoarthritis caused by ligament disruption has led to the research and development of biomimetic scaffolds combined with cell-based regeneration techniques. Current clinical focal defect repair strategies have had limited success. New scaffold-based approaches may provide solutions that can repair extensive damage and prevent osteoarthritis. This study utilized a novel scaffold design that accommodated strain gauges for shear and axial load monitoring in the canine stifle joint through implantable telemetry technology. Loading changes induced by ligament disruption are widely implicated in the development of injury-related osteoarthritis. Seeding the scaffold end with progenitor cells resulted in higher shear stress than without cell seeding and histology showed significantly more bone and cartilage formation. Biomechanically, the effect of transecting the anterior cruciate ligament was a significant reduction in braking load in shear, but no change axially, and conversely a significant reduction in push-off load axially, but no change in shear. This is the first study to report shear loads measured directly in knee joint tissue. Further, advances of these measurement techniques are critical to developing improved regeneration strategies and personalizing reliable rehabilitation protocols.

Co-culture of adipose derived stem cells and chondrocytes with surface modifying proteins induces enhanced cartilage tissue formation,
Waters, HA, Geffre CP, Gonzales DA, Grana WA, and Szivek JA
J Invest Surg, vol. 26, issue 3, pp. 118-26, PMID: 23514058, June 2013

Current treatments for focal cartilage defects include osteochondral allograft transplants—a common treatment for large defects and revisions of previously autografted joints. Allografts with weak osseous regions are usable, since bone remodeling replaces inferior quality bone. However, poor quality chondral surfaces on grafts preclude their use, leading to grafting material shortages. Endogenous adult stem cells can make hyalinelike cartilage tissue on scaffolds. To increase the number of usable allografts, tissue culture methods using adipose derived stem cells (ASCs) were developed to grow cartilage on grafts. Methods: Co-cultures utilized living chondrocytes in host cartilage, modeling in vivo conditions, and ASCs seeded on the allografts. Sterilized allografts were treated with Poly-L-Lysine and ProNectin. Tissue growth was analyzed and quantified with histological techniques. Results and Conclusions: Monoculture experiments produced tenuous cartilage formation when proteins were utilized and allograft surfaces were perforated. Extensive tissue formation was observed with coculture and the presence of type II collagen was confirmed with immunohistochemistry. Results demonstrate that co-culture techniques offer a better means of growing tissue on allograft cartilage surfaces. Additionally, the use of proteins to facilitate surface attachment produced more tissue formation demonstrating that cell attachment is crucial when growing cartilage on allografts. Development of new culture techniques to evaluate treatment strategies will accelerate the rate at which cartilage procedures using endogenous cells are possible. This will increase the number of usable grafts and allow critical selection of grafts to fit specific surfaces increasing surgical success by returning the joint to its native structure.

Implantable Sensor Technology: From Research to Clinical Practice, 
Ledet EH, D’Lima D, Westerhoff P, Szivek JA, Wachs RA, Bergmann G., 
J American Academy of Orthopaedic Surgeons, Vol 20, 6, 383-392. June 2012.

Abstract: For decades, implantable sensors have been used in research to provide comprehensive understanding of the biomechanics of the human musculoskeletal system. These complex sensor systems have improved our understanding of the in vivo environment by yielding in vivo measurements of force, torque, pressure, and temperature. Historically, implants have been modified to be used as vehicles for sensors and telemetry systems. Recently, microfabrication and nanofabrication technology have sufficiently evolved that wireless, passive sensor systems can be incorporated into implants or tissue with minimal or no modification to the host implant. At the same time, sensor technology costs per unit have become less expensive, providing opportunities for use in daily clinical practice. Although diagnostic implantable sensors can be used clinically without significant increases in expense or surgical time, to date, orthopaedic smart implants have been used exclusively as research tools. These implantable sensors can facilitate personalized medicine by providing exquisitely accurate in vivo data unique to each patient.

Mechanical loading of adipose derived stromal cells causes cell alignment,
DA Gonzales, AS Ferng, GP Geffre, JL Borg, M Miller, JA Szivek, J Biomedical Science and Engineering, 2011, 4, 355-359, JBiSE doi:10.4236/jbise.2011.45045

Abstract: Osteoarthritis is a debilitating disease that affects hundreds of millions of people worldwide. Current research involving growth and characterization of adipose derived stromal cells (ADSC) in vitro offers a potential solution for the treatment of cartilage defects that will allow patients to return to the physical activities they were involved in. Studies have shown that fibroblast cells grown in vitro respond to cyclic mechanical stretching by orienting in a direction perpendicular to the direction of stretch. ADSCs were isolated from human peripatellar adipose tissue dis- cards. Cells were cultured until confluent and seeded at a density of approximately 105 cells in silicone wells pretreated with ProNectin-F Plus. After stretching, relative alignment of the cells was ascertained using imaging software. Stretching cells for 3, 4, 8 and 12 hours resulted in noticeable cellular alignment of approximately 60 degrees relative to the direction of loading. Cell alignment is crucial for developing tis- sue-engineered cartilage that has similar mechanical properties to native cartilage. Mechanically loading cells is one method to achieve cell alignment. Since cell differentiation will be initiated after alignment, the resulting chondrocytes will be aligned, leading to organized collagen formation and resulting in a hyaline-like cartilage structure.

Load-Bearing at the Meniscofemoral Joint: An in vitro Study in the Canine Knee,
Pier Francesco Indelli, John A Szivek, Andrew Schnepp, William A Grana
The Duke Orthopaedic Journal, July 2010-June 2011;1(1):39-43

Abstract: Background: The role of the menisci on tibial load transmission and stress distribution has been extensively studied, but few studies have focused on the meniscofemoral joint during physiologic weightbearing. The objective of this study was to determine the contact areas and local contact stresses at the meniscofemoral interface during physiologic range of motion and axial-loading in the canine knee and to determine the influence of a partial or total meniscectomy.
Methods: Both fresh-frozen knees of 3 hound-type canines were tested in a universal testing machine configured for an axial-load of 90- 120 N. Measurement of the contact area and the local contact stress were done at three different knee angles (30; 50; 70) and with both menisci intact, after partial meniscectomy, and after total meniscectomy. Pressure distribution was estimated by using pressure sensitive film inserted above the menisci.
Results: After partial meniscectomy, contact areas at 50° of knee flexion decreased approximately 25% on both femoral condyles, and local contact stress increased 30% on the medial femoral condyle but remained unchanged on the lateral. After total meniscectomy, contact areas at 50° of knee flexion decreased approximately 75% on both femoral condyles, and local contact stress increased approximately 60% on the medial compartment and 100% on the lateral compartment.
Conclusions: These data suggest that a conservative partial meniscectomy leaves the meniscus with an inferior weight distribution function; decreasing, but not canceling the protection on the femoral hyaline cartilage. A dramatic decrease of contact area followed by an increase of local contact stress was noted after a total meniscectomy. The clinical value of this study is to emphasize the biomechanical value of surgical procedures addressing the repair of damaged menisci.

Load Measurement Accuracy from Sensate Scaffolds with and without a Cartilage Surface,
CP Geffre, PR Finkbone, CL Bliss, DS Margolis, JA Szivek, J Investigative Surgery, 23, 156–162, DOI: 10.3109/08941939.2010.481006, 2010

Abstract: The use of “sensate” scaffolds covered with tissue-engineered cartilage has emerged as a possible treatment option for focal articular cartilage defects. The ability to monitor joint loading provides several benefits that can be useful in both clinical and research situations. Previous studies have shown that these scaffolds can accurately monitor in vivo joint loading during various activities. However, the effect that an articular cartilage layer or soft tissue overgrowth has on scaffold sensitivity has not been tested. Eight scaffolds were tested with cartilage samples taken from four hounds. Three strain gauges were attached to each scaffold and a servo hydraulics system was used to test sensitivity while the scaffold was in contact with cartilage, metal, or silicone surfaces. Strain gauge sensitivity was calculated from load and strain measurements collected during testing. There was no significant difference between the mean strain gauge sensitivities when the scaffolds were in contact with the different surfaces: cartilage 30.9 ± 16.2 με/N, metal 31.8 ± 18.6 με/N, and silicone 30.6 ± 12.3 με/N. These results indicate that “sensate” scaffolds can be calibrated and used to monitor load with the presence of an articular cartilage layer.

Evaluation Of Chitosan-Calcium Phosphate Bone Fillers, 
CP Geffre, J Ochoa, JA Szivek, DS Margolis, J Investigative Surgery, 23, 134–141, DOI: 10.3109/08941930903564100, 2010.

Abstract: There are has been recent interest in utilizing calcium phosphate ceramics for treating bone defects because of the limitation associated with autografts and allografts. However, calcium phosphates are limited by long setting times, inappropriate mechanical properties, slow in vivo resorption and poor osteoinductivity. This has directed research towards finding a non- protein based compound, such as chitosan, to address these issues. The purpose of this study was to compare in vivo bone growth achieved using conventionally prepared chitosan-calcium phosphate bone filler to an extensively purified chitosan-calcium phosphate compound. Histomorphometry demonstrated a 290% increase in new bone formation when using the chitosan-calcium phosphate bone filler and a 172% increase with the purified chitosan-calcium phosphate compound compared to the increase seen with the untreated control group. This supports the applicability of chitosan-calcium phosphate bone fillers in the treatment of bone defects.

Novel Biomimetic Polymer Scaffold Design enhances Bone Ingrowth, 
CP Geffre, JA Szivek, JT Ruth, DW DeYoung, B Tellis, DS Margolis, J Biomed Mater Res 91A: 795–805, DOI: 10.1002/jbm.a.32251 JBMR-A, 2009.

Abstract: There has been recent interest in treating large bone defects with polymer scaffolds because current modalities such as autographs and allographs have limitations. Additionally, polymer scaffolds are utilized in tissue engineering applications to implant and anchor tissues in place, promoting integration with surrounding native tis- sue. In both applications, rapid and increased bone growth is crucial to the success of the implant. Recent studies have shown that mimicking native bone tissue morphology leads to increased osteoblastic phenotype and more rapid mineralization. The purpose of this study was to compare bone ingrowth into polymer scaffolds created with a biomimetic porous architecture to those with a simple porous design. The biomimetic architecture was de- signed from the inverse structure of native trabecular bone and manufactured using solid free form fabrication. Histology and microCT analysis demonstrated a 500–600% increase in bone growth into and adjacent to the biomimetic scaffold at five months post-op. This is in agreement with previous studies in which biomimetic approaches accelerated bone formation. It also supports the applicability of polymer scaffolds for the treatment of large tissue defects when implanting tissue-engineering constructs.

Phenotypic Characteristics of Bone in Carbonic Anhydrase II Deficient Mice
David S. Margolis, John A. Szivek, Li-Wen Lai, and Yeong-Hau H. Lien
Calcif Tissue Int Vol 82– 1, 66-76, 2008, DOI 10.1007/s00223-007-9098-x

Abstract:  Carbonic Anhydrase II (CAII) deficient mice were created to study the syndrome of CAII deficiency in human including osteopetrosis, renal tubular acidosis and cerebral calcification.  Although CAII mice have renal tubular acidosis, studies that analyzed only cortical bones of CAII mice found no changes characteristic of osteopetrosis.  Consistent with previous studies, the tibiae of CAII mice were significantly smaller than wild type (WT) mice (28.7 ± 0.9 mg vs. 43.6 ± 3.7 mg, p < 0.005) and the normalized cortical bone volume of CAII deficient mice (79.3 ± 2.2%) was maintained within 5% of WT mice (82.7 ± 2.3%, p < 0.05), however, metaphyseal widening of the tibial plateau was noted in CAII deficient mice that is consistent with osteopetrosis.  In contrast to cortical bone, trabecular bone volume demonstrated a nearly 50% increase in CAII deficient mice (22.9 ± 3.5% in CAII, compared to 15.3 ± 1.6% in WT, p < 0.001). In addition, histomorphometry demonstrated that bone formation rate was decreased by 68% in cortical bone (4.77 ± 1.65 mm3/mm2/day in WT vs. 2.07 ± 1.71 mm3/mm2/day in CAII mice, p < 0.05) and 55% in trabecular bone (0.617 ± 0.230 mm3/mm2/day in WT vs. 0.272 ± 0.114 mm3/mm2/day in CAII mice, p < 0.05) in CAII deficient mice.  The number of osteoclast was significantly increased in CAII deficient mice.The metaphyseal widening and changes in the trabecular bone are consistent with osteopetrosis, making the CAII deficient mouse a valuable model of human disease.

Sensate Scaffolds Coupled to Telemetry Can Monitor In Vivo Loading From Within a Joint Over Extended Periods of Time
Chris P. Geffre, Cody L. Bliss, John A. Szivek, Donald W. DeYoung, John T. Ruth, David S. Margolis
J Biomed Mater Res Part B: Appl Biomater 84B: 263–270, 2008

Abstract: Polymer scaffolds have been used as a tool to provide growth and integration of engineered tissue substrates to repair damaged tissues in many organ systems including articular cartilage. Previous work has shown that ‘‘sensate’’ scaffolds, with integrated strain gauges have the potential for use as both a delivery vehicle for engineered cartilage as well as a device that can measure real time, in vivo joint loading. The purpose of this study was to use an implanted subminiature telemetry system to collect in vivo joint loading measurements over an extended period following placement of a ‘‘sensate’’ scaffold. Measurements were collected from seven of nine sensors that were implanted into the stifles of three canines. The limb loading rates and load distribution through gait were dependent on stride time but did not vary with time post op. The peak loads were not dependent on stride time but significantly increased with time post op. This demonstrated that peak loading measured with ‘‘sensate’’ scaffolds can be used to monitor healing. The portability of the ‘‘sensate’’ scaffolds coupled to telemetry systems highlights the potential use of this system in a clinical research setting to gather important information to improve tissue engineering and rehabilitation regimens.

A Handheld Computer as Part of a Portable In Vivo Knee Joint Load-Monitoring System
JA Szivek, VS Nandakumar, CP Geffre, C. P. Townsend
Journal of Medical Devices, Vol. 2 / 035001-1 035001-2 / Vol. 2, Sept 2008 Trans of ASME DOI: 10.1115/1.2952815

Abstract: In vivo measurement of loads and pressures acting on articular cartilage in the knee joint during various activities and rehabilitative therapies following focal defect repair will provide a means of designing activities that encourage faster and more complete healing of focal defects. It was the goal of this study to develop a totally portable monitoring system that could be used during various activities and allow continuous monitoring of forces acting on the knee. In order to make the monitoring system portable, a handheld computer with custom software, a USB powered miniature wireless receiver, and a battery-powered coil were developed to replace a currently used computer, ac powered bench top receiver, and power supply. A Dell handheld running Windows Mo- bile operating system programed using LABVIEW was used to collect strain measurements. Measurements collected by the handheld-based system connected to the miniature wireless receiver were compared with the measurements collected by a hard- wired system and a computer based system during bench top testing and in vivo testing. The newly developed handheld-based system had a maximum accuracy of 99% when compared to the computer-based system.

Trabecular scaffolds created using micro CT guided fused deposition modeling
BC Tellis, JA Szivek, CL Bliss, DS Margolis, RK Vaidyanathan b, P. Calvert
Materials Science and Engineering C (2007), doi:10.1016/j.msec.2006.11.010

Abstract: Free form fabrication and high resolution imaging techniques enable the creation of biomimetic tissue engineering scaffolds. A 3D CAD model of canine trabecular bone was produced via micro CT and exported to a fused deposition modeler, to produce polybutylene terephthalate (PBT) trabeculated scaffolds and four other scaffold groups of varying pore structures. The five scaffold groups were divided into subgroups (n = 6) and compression tested at two load rates (49 N/s and 294 N/s). Two groups were soaked in a 25 °C saline solution for 7 days before compression testing. Micro CT was used to compare porosity, connectivity density, and trabecular separation of each scaffold type to a canine trabecular bone sample. At 49 N/s the dry trabecular scaffolds had a compressive stiffness of 4.94 ± 1.19 MPa, similar to the simple linear small pore scaffolds and significantly more stiff (p < 0.05) than either of the complex interconnected pore scaffolds. At 294 N/s, the compressive stiffness values for all five groups roughly doubled. Soaking in saline had an insignificant effect on stiffness. The trabecular scaffolds matched bone samples in porosity; however, achieving physiologic connectivity density and trabecular separation will require further refining of scaffold processing.

Sensate Scaffolds Can Reliably Detect Joint Loading
CL Bliss, JA Szivek, BC Tellis, DS Margolis, AB Schnepp, JT Ruth
J Biomed Mater Res Part B: Appl Biomater 81B: 30–39, 2007

Abstract: Treatment of cartilage defects is essential to the prevention of osteoarthritis. Scaffold-based cartilage tissue engineering shows promise as a viable technique to treat focal defects. Added functionality can be achieved by incorporating strain gauges into scaffolds, thereby providing a real-time diagnostic measurement of joint loading. Strain-gauged scaffolds were placed into the medial femoral condyles of 14 adult canine knees and bench top tested. Loads between 75 and 130 N were applied to the stifle joints at 308, 508, and 708 of flexion. Strain-gauged scaffolds were able to reliably assess joint loading at all applied flexion angles and loads. Pressure sensitive films were used to determine joint surface pressures during loading and to assess the effect of scaffold placement on joint pressures. A comparison of peak pressures in control knees and joints with implanted scaffolds, as well as a comparison of pressures before and after scaffold placement, showed that strain-gauged scaffold implantation did not significantly alter joint pressures. Future studies could possibly use strain-gauged scaffolds to clinically establish normal joint loads and to determine loads that are damaging to both healthy and tissue-engineered cartilage. Strain-gauged scaffolds may significantly aid the development of a functional engineered cartilage tissue substitute as well as provide insight into the native environment of cartilage.

Selective cell proliferation can be controlled with CPC particle coatings 
JA Szivek, DS Margolis, AB Schnepp, WA Grana, SK Williams 
J Biomed Mater Res 81A: 939–947, 2007

Abstract: To develop implantable, engineered, cartilage constructs supported by a scaffold, techniques to encourage rapid tissue growth into, and on the scaffold are essential. Preliminary studies indicated that human endothelial cells proliferated at different rates on different calcium phosphate ceramic (CPC) particles. Judicious selection of particles may encourage specific cell proliferation, leading to an ordered growth of tissues for angiogenesis, osteogenesis, and chondrogenesis. The goal of this study was to identify CPC surfaces that encourage bone and vascular cell growth, and other surfaces that support chondrocyte growth while inhibiting proliferation of vascular cells.  Differences in bone and vascular cell proliferation were observed when using epoxy without embedded CPCs to encourage bone cells, and when three CPCs were tested, which encouraged vascular cell proliferation. One of these (CPC 7) also substantially depressed cartilage cell proliferation. Only one small-diameter crystalline CPC (CPC 2) supported rapid chondrocyte proliferation, and maintained the cartilage cell phenotype.

A Comparison of the Effects of Radiofrequency Treatment and Mechanical Shaving for Meniscectomy 
William A. Grana, John A. Szivek, Andrew B. Schnepp, and Renata Ramos,
Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 22, No 8 (August), 2006: pp 884-888

Abstract: Purpose:The goal of this ex vivo pilot study was to compare radiofrequency treatment with cutting and shaving treatment of meniscal tears by use of a mechanical testing procedure and electron microscopy to establish the mechanical characteristics and qualitative appearance of meniscal tissue after the use of each of these procedures. Methods:In this study 136 menisci were explanted and divided into 4 groups: a damaged, untreated control group; a group damaged in the same way as the control group and treated by mechanical shaving of the meniscal tear; a group damaged in a similar way and then treated by radiofrequency by use of a radiofrequency wand; and a fourth group in which plunge-cutting by use of the radiofrequency wand was used to resect the tissue, beginning at the superior surface of the meniscus in a place that corresponded to the location of the meniscal tears. The menisci were then tested for strength by applying radial tension to the tear. Electron microscopy at low and high magnification was used to evaluate the appearance of the surface of the menisci after shaving or radiofrequency treatment. Results:Static mechanical testing to failure showed no significant difference between the control group and the 3 test groups. However, there was a statistically significant difference between the radiofrequency-treated groups and the mechanically shaved group at the .033 level. On fatigue testing, there was no statistically significant difference in the failure cycles, but the coefficient of variation was 8 times greater for the mechanically shaved menisci versus the radio- frequency-treated menisci. Scanning electron microscopy showed that the mechanically treated menisci had flat surfaces with clefts or fissures. The radiofrequency-treated menisci had a homogeneous appearance without clefts. Conclusions:This study showed that radiofrequency-treated damaged tissue leaves a qualitatively different surface from the mechanically treated menisci, which failed at a significantly higher load on static testing. On fatigue testing, there was greater variation in the number of cycles to failure of mechanically treated specimens versus the radiofrequency-treated menisci. Clinical Relevance:Although recurrent meniscal tears are uncommon, they may be of value in evaluating different methods of meniscectomy. This study points out mechanical and qualitative differences between shaved and radiofrequency-treated meniscectomy.

An Instrumented Scaffold can Monitor Loading in the Knee Joint, 
JA Szivek, CL Bliss, CP Geffre, DS Margolis, DW DeYoung, JT Ruth, AB Schnepp, BC Tellis, RK Vaidyanathan, J Biomed Mater Res - B, 79B, 218-228, 2006.

Abstract: No technique has been consistently successful in the repair of large focal defects in cartilage, particularly in older patients. Tissue engineered cartilage grown on synt hetic scaffolds with appropriate mechanical properties will provide an implant which could be used to treat this problem. A means of monitoring loads and pressures acting on cartilage, at the defect site, will provide information needed to understand inte gration and survival of engineered tissues. It will also provide a means of evaluating rehabilitation protocols. A "sensate" scaffold with calibrated strain sensors attached to its surface, combined with a subminiature radio transmitte r, was developed and utilized to measure loads andpressures during gait. In an animal study utilizing six dogs, peak loads of 120 N and peak pressures of 11 MPa were measured during relaxed gait. Ingrowth into the scaffold characterized after 6 months in vivo indicated it was well anchored and bone formation was continuing. Cartilage tissue formation was noted at the edges of the defect at the joint-scaffold interfaces. This suggested that native cartilage integration in future formulations of this scaffo ld configured with engineered cartilage, will be a possibility.

In Vivo Strain Measurements from Hardware and Lamina during Spine Fusion, 
John A. Szivek, Rolando F. Roberto , Journal Biomedical Materials 
Research-B 75B:243-250, 2005.

Abstract: Currently, spine fusion is determined using radiography and clinical evaluation. There are discrepancies between radiographic evidence and direct measurements of fusion, such as operative exploration and biomechanical or histological measurements. In order to facilitate the rapid return of patients to normal activities, a monitoring technique to accurately detect fusion in vivo and to prevent overload during the postoperative period would be useful. The objectives of this study were to develop an implantable monitoring system consisting of CPC-coated strain gauges and a radio transmitter to detect the onset of fusion and measure strain during postsurgical activi ties. A patient underwent anterior release and fusion, followed by posterior instrumentation and fusion with segmental spinal instrumentation. Four strain gauges were placed during surgery. One was attached to the left-side rod and one to each of the lami na at T9, T10, and T11. An externally powered implanted radio transmitter attached to the gauges was placed in a subcutaneous pouch. Strains were monitored weekly and tabulated during various activities for 7 months. Peak strains during twisting and bendi ng were tabulated to detect the onset of fusion. Strains were also recorded during activities such as climbing off an examination table, rising from a chair, and climbing stairs. Strains collected from the left rod indicated that, immediately postoperativ ely, it was loaded at acceptable levels. The largest and most consistent strain changes measured from the lamina were recorded during twisting.

Functionally improved bone in Calbindin-D28k knockout mice
David S. Margolis, John A. Szivek, Li-Wen Lai, and Yeong-Hau H. Lien 
Bone 39 (2006) 477–484

Abstract: In vitro studies indicate that calbindin-D28k, a calcium binding protein, is important in regulating the life span of osteoblasts as well as the mineralization o f bone extracellular matrix. The recent creation of a calbindin-D28k knockout mouse has provided the opportunity to study the physiological effects of the protein on bone remodeling in vivo. In this experiment, quantitative histomorphometry was u sed to characterize bone remodeling in normal and calbindin-D28k knockout mice. Cantilever bend testing was used to characterize the stiffness, failure loads and the elastic moduli of femora from normal and knockout mice. Statistical significance was dete rmined using ANOVA for all histology and histomorphometry parameters, and significance was determined using a dependent t-test for all biomechanical properties. The calbindin-D28k knockout mice had significantly decreased mineral apposition rates and bone formation rates. Despite the lower bone formation rates, the bone volume was larger in knockout mice indicating decreased bone resorption rates and less dynamic bone remodeling. The mechanical testing demonstrated that the bones of knockout mice are stif fer and fail at higher loads than the bones of normal mice; however the elastic moduli of the bones in both mice are similar. This indicates that the increased stiffness and failure load were a result of the increased bone volume. The calbindin-D28k knock out mouse provides a good model to study how vitamin D and calcium binding proteins regulate bone modeling and remodeling in vivo.

TGF-ß1 Accelerates Bone Growth Into Porous TCP coated Scaffolds 
John A. Szivek,David Margolis, Bijiibaa' K. Garrison, Ehren Nelson, Ranji K. Vaidyanathan, Don W. DeYoung, Journal Biomedical Materials Research-B, Vol: 73B-1, 43-53, 2004.

Abstract: Porous polybutylene terephthalate (PBT) scaffold systems were tested as orthopedic implants to determine whether these scaffolds could be used to detect strain transfer following bone growth into the scaffold. Three types of scaffold systems were tested: porous PBT scaffolds, porous PBT scaffolds with a thin -tricalcium phosphate coating (LC-PBT), and porous PBT scaffolds with the TCP coating vacuum packed into the scaffold pores (VI-PBT). In addition, the effect of applying TGF- 1 to scaffolds as an enhancement was examined. The scaffolds were placed onto the femora of rats and left in vivo for 4 months. The amount of bone ingrowth and the strain transfer through variou s scaffolds was evaluated by using scanning electron microscopy, histology, histomorphometry, and cantilever bend testing. The VI-PBT scaffold showed the highest and most consistent degree of mechanical interaction between bone and scaffold, providing str ain transfers of 68.5% ( 20.6) and 79.2% ( 8.7) of control scaffolds in tension and compression, respectively. The strain transfer through the VI-PBT scaffold decreased to 29.1% ( 24.3) and 30.4% ( 25.8) in tension and compression when used with TGF- 1. T GF- 1 enhancement increased the strain transfer through LC-PBT scaffolds in compression from 9.4% ( 8.7) to 49.7% ( 31.0). The significant changes in mechanical strain transfer through LC-PBT and VI-PBT scaffolds correlated with changes in bone ingrowth f raction, which was increased by 39.6% in LC-PBT scaffolds and was decreased 21.3% in VI-PBT scaffolds after TGF- 1 enhancement. Overall, the results indicate that strain transfer through TCP-coated PBT scaffolds correlate with bone ingrowth after implanta tion, making these instrumented scaffolds useful for monitoring bone growth by monitoring strain transfer.

Bilateral Symmetry of Biomechanical Properties in Mouse Femora 
David S. Margolis, Yeong-Hau H. Lien, Li-Wen Lai, and John A. Szivek 
Medical Engineering and Physics, Vol: 26-4, 349-353 , 2004.

Abstract: Bone healing and remodeling are commonly examined in animal models by comparing one femur (experimental) to the contralateral femur (control) with the assumption that they are id entical with respect to their biomechanical properties. While past studies have characterized the symmetry in geometrical properties in many types of animal bones, few studies have compared the symmetry in the biomechanical properties. The purpose of this study was to determine whether there is symmetry in the mechanical properties of mouse femora. Strain gauges were attached to the posterior surface of the femora of C57BL/6 mice, parallel to the long axis of the bone. The femora were mechanically tested in cantilever bending while strain values were recorded. Moments of inertia, cortical areas, and moduli of elasticity were determined from strains and cross-sectional properties. Mouse femora demonstrated an average strain difference of 0.4% in tension an d 1.4% in compression. Elastic moduli differed by 6.6% and 0.9% in tension and compression, respectively, and failure strength differed by an average of 2.0%. Statistical analysis showed there were no significant differences in strain, modulus, or failure load values for the mice, indicating mechanical and geometrical symmetry of mouse femora in cantilever bending.

TGF-ß1 Accelerates Bone Bonding to a Blended CPC Coating: A Dose Response Study,
John A. Szivek, Ehren R. Nelson, Steven D. Hajdu, Kelly Yablonski, Don W. DeYoung Journal Biomedical Materials Research-A 68A-3, 537-543, 2004.

Abstract: In vivo strain measurements can facilitate the study of the bone remodeling response to loading and load changes. Calcium phosphate ceramic (CPC) coatings have b een used to attach strain gauges to bone for extended periods of time, but require up to 12 weeks for adequate CPC-to-bone bonding. Transforming growth factor-(TGF)-1, an osteoinductive growth factor, was used as a surface enhancement to accelerate bone g rowth and bonding to CPC particles. The aim of this study was to find an optimal dosage of TGF- 1 to accelerate the attachment process. CPCcoated strain gauges were enhanced with doses of 0.5, 1.0, or 2.0 g of TGF- 1 per gauge. Gauges were placed on the f emora of dogs, which were exercised daily and fed ad libitum. After 3, 6, and 12 weeks, gauge attachment was quantitatively assessed using mechanical testing and histomorphometry. Gauge attachment was also qualitatively assessed using back scatte r electron microscopy. Agreement of the mechanical test results with both the back scatter electron microscopy images and histomorphometry results showed that the 1.0 g per gauge dose of TGF- 1 is an optimal dose to accelerate bone formation and attachmen t to CPC-coated strain gauges.

Evaluation of a new CPC-to-Gauge Bonding Technique with the use of In Vitro Fuid Flow 
Mark M. Fernandez, John A. Szivek, David S. Margolis, 
Journal of Biomedical Materials Research – B Vol: 66B, 514-519, 2003.

Abstract: Strain gauging enables the measurement of bone deformation during physical activity, leading to a better understanding of the physiological effects of loading on bone gr owth and remodeling. Development of a technology that will withstand long-term in vivo exposure and bond securely to bone is imperative for accurate, consistent measurement collection. Polysulfone is currently used to attach calcium-phosphate cer amic (CPC) particles, which promote bone-to-gauge bonding, to polyimide-backed strain gauges. This study evaluated the use of an implant-grade epoxy as an alternative CPC–polyimide adhesive. Polyimide–epoxy–CPC interfaces wer e loaded to failure and shear strengths calculated. In vitro studies providing a constant flow of medium over test specimens were designed, and long-term in vitro fluid exposure studies of the epoxy's shear strength were conduc ted. Average shear strength of polysulfone-polyimide interfaces were reported to be 7 MPa.11 The average shear strength of the epoxy-polyimide interface before long-term in vitro exposure was 17 MPa, which is stronger than the shear strength of t he bone–CPC interface.12 The strength of the epoxy–polyimide interface decreased to 6.8 MPa after 24 weeks in vitro and 3 MPa after 24 weeks in vivo.

The Effect of Implant Overlap on the Mechanical Properties of the Femur, 
Harris T, Ruth JT, Szivek J, Haywood B., J Trauma; 54(5):930-935, 2003.

Abstract: There exists a clinical dilemma in the treatment of supracondylar femur fractures distal to compression hip screw (CHS) devices. Retrograde femoral nails have shown compa rable clinical outcomes to fixed angle plate devices in the treatment of supracondylar femur fractures. FN The most biomechanically stable relationship between the side plate of a CHS and retrograde intramedullary (IM) femoral nail is not well defined. Th is becomes a technical issue when treating supracondylar femur fractures with a retrograde nail in patients with a history of CHS fixation of an intertrochanteric fracture. The proximal end of the nail and the interlocking screws may act as a stress riser in the femoral diaphysis and fracture at the proximal end of retrograde femoral nail is a described complication. FN We hypothesized that implant overlap would minimze regional strain and increase failure strength in the femoral diaphysis. The purpose of this study was to determine how the strength of the bone-implant interface changed with different amounts of implant overlap between a CHS plate and statically locked retrograde femoral nail.

An Implantable Strain Measurement System Designed to Detect Spine Fusion: Preliminary Results from a Biomechanical and In Vivo Study, 
J. A. Szivek, R. Roberto, J. M. Slack, B. S. M. Majeed, SPINE, 27-5, 487-497, 2002.

Abstract: The strain distribution on the thoracic vertebrae during anteroposterior bending and torsion was examined for use with an implantable strain gauge system and miniature radio transmitter to identify strain gauge placement sites by testing cadaver spines, and to evaluate an implantable gauge bonding technique and subminiature radio transmitter for accurate strain monitoring.

Fusion is determined currently through the use of radiographic techniques. Discrepancies exist between radiographic evidence and more direct measurements of fusion such as operative exploration an d biomechanical or histologic measurements. To facilitate the return of patients to full unrestricted activity, it would be useful to develop a technique for accurate in vivo determination of fusion. Three cadaver spines were tested during antero posterior bending and torsional loading in the control, instrumented, and instrumented plus polymethylmethacrylate states. The spines were instrumented with an ISOLA construct, and a simulated fusion was achieved through the application of polymethylmetha crylate. Strain gauges were attached in uniaxial, biaxial, and rosette configurations. The principal strains were calculated. Calcium phosphate ceramic-coated gauges were implanted in patients and recovered after up to 15 months in vivo. A radio transmitter was developed and tested for use in patients. The largest and most consistent strain changes after simulated fusion were recorded during torsional loading on the laminae of a vertebra directly underneath a hook. Calcium phosphate ceramic-coate d strain gauges showed excellent bone bonding to the lamina when fusion occurred. Radio telemetry accurately tracked strain magnitudes and strain rates expected in patients. The consistency obtained in torsional loading indicates that this type of loading will provide the most useful data from patients in vivo. Excellent bonebonding and accurate strain transmission using a longterm strain measurement system and miniature radio transmitter indicate that strains collected from patients with this sy stem will be accurate.

Linear And Volumetric Wear of Tibial Polyethylene Retrievals From PCL Retaining Knee Arthroplasties 
James Benjamin, John A Szivek, Gabriel Dersam, Sara Persselin, Randon Johnson, Clinical Orthopaedics and Related Research, 392, 131-138, 2001.

Abstract: Linear and volumetric wear was measured in 33 tibial polyethylene inserts from three different cruciate-retaining knee systems retrieved at the time of revision surgery. Wear patterns also were evaluated and classified. Eccentric and asymmetric wear patterns were seen in 78% of inserts with flat articulating geometry versus 12% in inserts with curved anteroposterior geometry. The mean lin ear wear rate was .35 mm/year (range, .05–1.68 mm/year) and the mean volumetric wear rate was 794 mm3/year (range, 24–4088 mm3/year). Linear and volumetric wear rates showed a negative correlation with the length of implantation. Linea r wear rates also showed a negative correlation with patient weight.

Long-Term Measurement of Bone Strain In Vivo: The Rat Tibia
B.A. Rabkin, J.A.Szivek, J. Schonfeld, B.P. Halloran,JBMR (Applied Biomaterials) Vol 58: 3, ,277-281, 2001.

Abstract: Despite the importance of strain in regulating bone metabolism, knowledge of strains induced in bone in vivo during normal activities is limited to short-term s tudies. Biodegeneration of the bond between gauge and bone is the principle cause of this limitation. To overcome the problem of bond degeneration, a unique calcium phosphate ceramic (CPC) coating has been developed that permits long-term attachment of mi crominiature strain gauges to bone. Using this technique, we report the first long-term measurements of bone strain in the rat tibia. Gauges, mounted on the tibia, achieved peak or near peak bonding at 7 weeks. Measurements were made between 7–1 0 weeks. Using ambulation on a treadmill, the pattern and magnitude of strain measured in the tibia remained relatively constant between 7–10 weeks post implantation. That strain levels were similar at 7 and 10 weeks suggests that gauge bonding is stable. These data demonstrate that CPC-coated strain gauges can be used to accurately measure bone strain for extended periods, and provide an in vivo assessment of tibial strain levels during normal ambulation in the rat.

Strain transfer between a CPC coated strain gauge and cortical bone during bending 
N. M. Cordaro, J. A. Weiss, J. A. Szivek , JBMR (Applied Biomaterials), 58:2, 147-155, 2001.

Abstract: The finite element method was used to simulate strain transfer from bone to a calciumphosphate ceramic (CPC) coated strain gauge. The model was constructed using gross morphometric and histological measurements obtained from previou s experimental studies. Material properties were assigned based on experiments and information fromthe literature. Boundary conditions simulated experimental cantilever loading of rat femora. The model was validated using analytical solutions based on the theory of elasticity as well as direct comparison to experimental data obtained in a separate study. The interface between the bone and strain gauge sensing surface consisted of layers of polysulfone, polysulfone/CPC, and CPC/ bone. Parameter studies exa mined the effect of interface thickness and modulus, gauge geometry, partial gauge debonding, and waterproofing on the strain transfer fromthe bone to the gauge sensing element. Results demonstrated that interface thickness and modulus have a significant effect on strain transfer. Optimal strain transfer was achieved for an interface modulus of approximately 2 GPa. Strain transfer decreased consistently with increasing interface thickness. Debonding along the lateral edges of the gauge had little effect, while debonding proximal and distal to the sensing element decreased strain transfer. A waterproofing layer decreased strain transfer, and this effect was more pronounced as the modulus or thickness of the layer increased. Based on these simulations, spec ific recommendations were made to optimize strain transfer between bone and CPC coated gauges for experimental studies.

Surface Enhancements Accelerate Bone Bonding to CPC Coated Strain Gauges, 
N. Cordaro, J. A. Szivek , D. W. DeYoung,, J. Biomedical Materials Research, Vol 56:1 109-119, 2001.

Abstract: Calcium phosphate ceramic (CPC)-coated strain gauges have been used for in vivo bone strain measurements for up to 18 weeks, but they require 6 to 9 weeks for s ufficient bonding. Osteogenic protein-1 (OP-1), PepTite™ (a proprietary ligand), calcium sulfate dihydrate (CSD), transforming growth factor b-1 (TGF-b1 ), and an endothelial cell layer with and without TGF-b1 were used as surface enhancements to acc elerate bone-to-CPC bonding. Young male Sprague–Dawley rats were implanted with unenhanced and enhanced CPC-coated gauges. Animals were allowed normal activity for 3 weeks and then calcein labeled. Femurs were explanted following euthanasia. A g auge was attached with cyanoacrylate to the opposite femur in the same position as the CPC-coated gauge. Bones were cantilever-loaded to assess strain transfer. They were sectioned and stained with mineralized bone stain (MIBS) and examined with transmitt ed and ultraviolet light. Mechanical testing indicated increased sensing accuracy for TGF-b1 and OP-1 enhancements to 105 ± 14% and 92 ± 12% versus 52 ± 44% for the unenhanced gauges. The PepTite™ and the endothelialcell-layer-enhanced gauges showed lower sensing accuracy, and histology revealed a vascular layer near CPC particles. TGF-b1 increased bone formation when used prior to endothelial cell sodding. CSD prevented strain transfer to the femur. TGF-b1 and OP-1 surface enhancements produced accu rate in vivo strain sensing on the rat femur after 3 weeks.

A Comparison of In Vitro and In Vivo Degradation of Two CPC Strain Gauge Coatings, 
J.A. Szivek, A.G. Battraw, S. Perrselin, P.L. Anderson, JBMR (Applied Biomaterials), Vol 53:3, 211-215, 2000. Cover Article,

Abstract: Calcium phosphate ceramic (CPC) coated strain gauges have been used to measure bone strain in animal models for up to 16 weeks and are being developed to collect measu rements in patients for periods of 1 year or more. A published surface roughening and heat treating procedure produced improved dry strength and in vivo stability of CPC-gauge interfaces after 16 weeks. The long term bond strength of two CPC-gaug e interfaces prepared using the roughening and heat treating process were evaluated after up to 1 year in vitro and in vivo using a lap shear test. The feasibility of using an in vitro test to predict long term in vivo interface changes was established. A blended tricalcium phosphate 1 hydroxyapatite had a CPC-gauge interface strength which decreased from 6.07 ± 2.64 MPa at 16 weeks to 4.71 ± 1.840 MPa after 1 year in Hanks Balanced Salts (HBS). The same coating had a s trength that decreased from 8.51 ± 2.63 MPa at 16 weeks to 5.35 ± 1 MPa after 1 year in vivo. A soluble calcium enhanced hydroxyapatite had an interface strength of 4.83 ± 1.106 MPa after 16 weeks and 4.516 1.100 MPa after 1 year in HBS. The same coating had an interface strength of 8.34 6 2.40 MPa after 16 weeks and 5.20 ± 2.00 MPa after 1 year in vivo. Although interface strengths decreased slightly with time in vivo, after 1 year they were in the same strength rangeas published CPC-bone interface strengths of 4.8 ± 2.4 MPa. Comparison of in vitro with in vivo results indicated that in vitro results were a good predictor of strength change in the blended CPC coating, but a poorer predictor of strength changes in the soluble calciumenhanced coating

The Effect of Lateral Muscle Loading on Femoral Strain Distributions in Bench top Hip Model 
J.A. Szivek, J. B. Benjamin, P. L. Anderson, Medical Engineering and Physics, 22, 109-116, 2000.

Abstract: Experimental models that have been used to evaluate hip loading and the effect of hip implants on bone often use only a head load and abductor load. Anatomic considerations and in vivo measurement s have lead several investigators to suggest that these models are inaccurate because they do not incorporate the loads imposed by additional muscles. The aim of this study was to evaluate the strains in the proximal and mid diaphysis of the femur for fiv e hip loading models, one with a head load and abductor load only and four which incorporated lateral muscle loads as well. Head load to body weight load ratios were used to evaluate the physiologic accuracy of these models and strains were compared to de termine the extent of strain changes as a function of model complexity. All models which incorporated additional lateral muscle loads more accurately simulated head load to body– weight load ratios than the simple abductor-only model. The model which incorporated a coupled vastus lateralis and iliotibial band load in addition to the abductor load provided the simplest configuration with a reasonable body–weight to head-load ratio.