Rameshwar (Ram) Rao MD PhD

All Publications

• POST-TRANSPLANT LYMPHOPROLIFERATIVE DISEASE IN A PATIENT WITH SCHIMKE IMMUNO-OSSEOUS DYSPLASIA Rao, R., Grimm, P., Lewis, D., Spunt, S., Marks, L. WILEY. 2023: S75-S76

  View details for Web of Science ID 001042987300147

• Leiomyomatosis in an Infant With a SUFU Splice Site Variant: Case Report. Journal of pediatric hematology/oncology Rao, R. R., Dulken, B. W., Matalon, D. R., Borensztein, M., McGuinness, M., Cizek, S. M., Bruzoni, M., Tan, S. Y., Kreimer, S. 2022

  Abstract

  Heterozygous loss-of-function variants in the suppressor of fused protein gene (SUFU) can result in Gorlin syndrome, which is characterized by an increased frequency of basal cell carcinoma, medulloblastoma, odontogenic keratocysts, as well as other tumors. We describe a case of a 5-month-old female who presented with multiple intra-abdominal leiomyomata and was found to have a likely pathogenic splice site variant in the SUFU gene. This is the first reported case of leiomyomatosis secondary to a pathogenic SUFU variant in an infant and may represent an early, atypical presentation of Gorlin syndrome.

  View details for DOI 10.1097/MPH.0000000000002454

  View details for PubMedID 35398865

• Ewing's Sarcoma in a Patient with Crohn's Disease Treated with Ustekinumab: A Case Report Journal of Adolescent and Young Adult Oncology Rao, R. R., Majlessipour, F., Ziring, D. A., Baca, N. M. 2020
• Mapping the Road to Recovery: Shorter Stays and Satisfied Patients in Posterior Spinal Fusion. Journal of pediatric orthopedics Rao, R. R., Hayes, M., Lewis, C., Hensinger, R. N., Farley, F. A., Li, Y., Caird, M. S. 2017; 37 (8): e536-e542

  Abstract

  Adolescent idiopathic scoliosis (AIS) patients undergoing posterior spinal fusion (PSF) experience variations in their hospital care, which may lead to differences in objective and patient-reported outcomes. The purpose of this study was to demonstrate that using plan of care-educating families preoperatively and standardizing some aspects of care-would decrease time to mobility and time to discharge while maintaining pain control and patient satisfaction.Chart review was conducted in 3 groups-preprotocol (December 2008 to December 2009, n=51), first protocol (December 2, 2009 to July 24, 2013, n=100), and second protocol (July 25, 2013 to June 1, 2014, n=39)-to track pain scores (0 to 10), time to regular diet, Foley catheter removal, epidural catheter removal, mobility, and discharge. Patient satisfaction surveys (0 to 10) were administered before discharge. Statistical analysis was performed using a 1-way analysis of variance test with Tukey post hoc analysis.Average pain scores were similar in all groups. Time to sitting was significantly reduced in both first protocol (27.2±9.8 h, P=1×10) and second protocol (28.4±13.6 h, P=3×10) compared with preprotocol (40.2±15.4 h). Time to discharge was significantly lower in second protocol (84.3±27.2 h, P=0.036) compared with first protocol (98.4±27.8 h). Patient satisfaction with care was significantly higher in first protocol (9.1/10, P=2×10) and second protocol (8.6/10, P=5×10) compared with preprotocol (6.5/10).By educating families preoperatively and standardizing portions of postoperative care in PSF for AIS, pain scores were significantly reduced while overall satisfaction remained high. Specifically, by removing the epidural and Foley catheters on postoperative day 2, time to discharge was dramatically decreased by 15 hours. The application of a multidisciplinary, evidence-driven plan of care for AIS patients undergoing PSF improves throughput and has beneficial effects on objective and patient-reported outcomes.Level III-retrospective case series.

  View details for DOI 10.1097/BPO.0000000000000773

  View details for PubMedID 27137901

• Treatment of Unicameral Bone Cysts of the Proximal Femur With Internal Fixation Lessens the Risk of Additional Surgery. Orthopedics Wilke, B., Houdek, M., Rao, R. R., Caird, M. S., Larson, A. N., Milbrandt, T. 2017; 40 (5): e862-e867

  Abstract

  Little data exist to guide the treatment of unicameral bone cysts in the proximal femur. Methods of treatment include corticosteroid injections, curettage and bone grafting, and internal fixation. The authors completed a multi-institutional, retrospective review to evaluate their experience with proximal femoral unicameral bone cysts. They posed the following questions: (1) Does internal fixation reduce the risk of further procedures for the treatment of a unicameral bone cyst? (2) Is radiographic healing faster with internal fixation? Following institutional review board approval, the authors conducted a retrospective review of 36 patients treated for a unicameral bone cyst of the proximal femur at their institutions between 1974 and 2014. Medical records and radiographs were reviewed to identify patient demographics and treatment outcomes. Tumor locations included femoral neck (n=13), intertrochanteric (n=16), and subtrochanteric (n=7). Initial treatment included steroid injection (n=2), curettage and bone grafting (n=9), and internal fixation with curettage and bone grafting (n=25). Mean time was 9 months to radiographic healing and 15 months to return to full activity. The number of patients requiring additional surgeries was increased among those who did not undergo internal fixation. There was no difference in time to radiographic healing. However, time to return to normal activities was reduced if patients had received internal fixation. A significant reduction in additional procedures was observed when patients had been treated with internal fixation. Although this did not influence time to radiographic healing, patients did return to normal activities sooner. Internal fixation should be considered in the treatment of proximal femoral unicameral bone cysts. [Orthopedics. 2017; 40(5):e862-e867.].

  View details for DOI 10.3928/01477447-20170810-01

  View details for PubMedID 28817159

• Ultrasound Utility in the Diagnosis of a Morel-Lavallée Lesion. Case reports in emergency medicine LaTulip, S., Rao, R. R., Sielaff, A., Theyyunni, N., Burkhardt, J. 2017; 2017: 3967587

  Abstract

  Morel-Lavallée lesions are uncommon injuries that can be associated with significant comorbidities if not detected early. Rapid diagnosis in the Emergency Department could significantly improve patient outcomes. We describe the diagnosis of such a lesion through the use of ultrasound imaging in the Emergency Department to utilize a fast, cost-effective imaging technique that does not subject the patient to radiation exposure. Our patient received surgical consultation but improved with conservative management. Ultrasound findings associated with this lesion do not require specialized equipment and should be considered when evaluating soft tissue lesions using point of care ultrasound.

  View details for DOI 10.1155/2017/3967587

  View details for PubMedID 28255470

  View details for PubMedCentralID PMC5309421

• Vocal Cord Paresis After Posterior Spinal Fusion to Treat Adolescent Idiopathic Scoliosis: A Case Report. JBJS case connector Rao, R. R., Ha, J., Farley, F. A., Koopmann, C. F., Caird, M. S. 2016; 6 (4): e97

  Abstract

  A 15-year-old girl with adolescent idiopathic scoliosis with a 50° curve underwent posterior spinal fusion from T3 to T11. After discharge from the hospital, the patient reported dysphonia and dysphagia. Flexible nasendoscopy confirmed left vocal cord paresis. Stretch injury to the recurrent laryngeal nerve from the left T5 pedicle screw or intubation may have caused the vocal cord paresis. The pedicle screw was removed during revision surgery. Postsurgically, the patient demonstrated immediate and ultimately full recovery and no longer had any symptoms.To our knowledge, this is the first case report of vocal cord paresis most likely caused by pedicle screw position after posterior spinal fusion.

  View details for DOI 10.2106/JBJS.CC.16.00090

  View details for PubMedID 29252751

• Collagen/fibrin microbeads as a delivery system for Ag-doped bioactive glass and DPSCs for potential applications in dentistry JOURNAL OF NON-CRYSTALLINE SOLIDS Chatzistavrou, X., Rao, R. R., Caldwell, D. J., Peterson, A. W., McAlpin, B., Wang, Y., Zheng, L., Fenno, J., Stegemann, J. P., Papagerakis, P. 2016; 432: 143–49

  View details for DOI 10.1016/j.jnoncrysol.2015.03.024

  View details for Web of Science ID 000367769100019

• Dual-phase osteogenic and vasculogenic engineered tissue for bone formation. Tissue engineering. Part A Rao, R. R., Vigen, M. L., Peterson, A. W., Caldwell, D. J., Putnam, A. J., Stegemann, J. P. 2015; 21 (3-4): 530-40

  Abstract

  Minimally invasive, injectable bone tissue engineering therapies offer the potential to facilitate orthopedic repair procedures, including in indications where enhanced bone regeneration is needed for complete healing. In this study, we developed a dual-phase tissue construct consisting of osteogenic (Osteo) and vasculogenic (Vasculo) components. A modular tissue engineering approach was used to create collagen/fibrin/hydroxyapatite (COL/FIB/HA) hydrogel microbeads containing embedded human bone marrow-derived mesenchymal stem cells (bmMSC). These microbeads were predifferentiated toward the osteogenic lineage in vitro for 14 days, and they were then embedded within a COL/FIB vasculogenic phase containing a coculture of undifferentiated bmMSC and human umbilical vein endothelial cells (HUVEC). In vitro studies demonstrated homogenous dispersion of microbeads within the outer phase, with endothelial network formation around the microbeads over 14 days in the coculture conditions. Subcutaneous injection into immunodeficient mice was used to investigate the ability of dual-phase (Osteo+Vasculo) and control (Osteo, Vasculo, Blank) constructs to form neovasculature and ectopic bone. Laser Doppler imaging demonstrated blood perfusion through all constructs at 1, 4, and 8 weeks postimplantation. Histological quantification of total vessel density showed no significant differences between the conditions. Microcomputed tomography indicated significantly higher ectopic bone volume (BV) in the Osteo condition at 4 weeks. At 8 weeks both the Osteo and Blank groups exhibited higher BV compared to the Vasculo and dual Osteo+Vasculo groups. These data not only show that osteogenic microbeads can be used to induce ectopic bone formation, but also suggest an inhibitory effect on BV when undifferentiated bmMSC and HUVEC were included in dual-phase constructs. This work may lead to improved methods for engineering vascularized bone tissue, and to injectable therapies for the treatment of orthopedic pathologies in which bone regeneration is delayed or prevented.

  View details for DOI 10.1089/ten.TEA.2013.0740

  View details for PubMedID 25228401

  View details for PubMedCentralID PMC4333512

• Vasculogenesis and Angiogenesis in Modular Collagen-Fibrin Microtissues. Biomaterials science Peterson, A. W., Caldwell, D. J., Rioja, A. Y., Rao, R. R., Putnam, A. J., Stegemann, J. P. 2014; 2 (10): 1497-1508

  Abstract

  The process of new blood vessel formation is critical in tissue development, remodeling and regeneration. Modular tissue engineering approaches have been developed to enable the bottom-up assembly of more complex tissues, including vascular networks. In this study, collagen-fibrin composite microbeads (100-300 μm in diameter) were fabricated using a water-in-oil emulsion technique. Human endothelial cells and human fibroblasts were embedded directly in the microbead matrix at the time of fabrication. Microbead populations were characterized and cultured for 14 days either as free-floating populations or embedded in a surrounding fibrin gel. The collagen-fibrin matrix efficiently entrapped cells and supported their viability and spreading. By 7 days in culture, endothelial cell networks were evident within microbeads, and these structures became more prominent by day 14. Fibroblasts co-localized with endothelial cells, suggesting a pericyte-like function, and laminin deposition indicated maturation of the vessel networks over time. Microbeads embedded in a fibrin gel immediately after fabrication showed the emergence of cells and the coalescence of vessel structures in the surrounding matrix by day 7. By day 14, inosculation of neighboring cords and prominent vessel structures were observed. Microbeads pre-cultured for 7 days prior to embedding in fibrin gave rise to vessel networks that emanated radially from the microbead by day 7, and developed into connected networks by day 14. Lumen formation in endothelial cell networks was confirmed using confocal sectioning. These data show that collagen-fibrin composite microbeads support vascular network formation. Microbeads embedded directly after fabrication emulated the process of vasculogenesis, while the branching and joining of vessels from pre-cultured microbeads resembled angiogenesis. This modular microtissue system has utility in studying the processes involved in new vessel formation, and may be developed into a therapy for the treatment of ischemic conditions.

  View details for DOI 10.1039/C4BM00141A

  View details for PubMedID 25177487

  View details for PubMedCentralID PMC4145346

• Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo. Acta biomaterialia Rao, R. R., Ceccarelli, J., Vigen, M. L., Gudur, M., Singh, R., Deng, C. X., Putnam, A. J., Stegemann, J. P. 2014; 10 (7): 3091-7

  Abstract

  Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0-20 mg ml(-1)), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml(-1) HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml(-1) HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 2 0mg ml(-1) HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.

  View details for DOI 10.1016/j.actbio.2014.03.010

  View details for PubMedID 24657675

  View details for PubMedCentralID PMC4041828

• Noninvasive quantification of in vitro osteoblastic differentiation in 3D engineered tissue constructs using spectral ultrasound imaging. PloS one Gudur, M. S., Rao, R. R., Peterson, A. W., Caldwell, D. J., Stegemann, J. P., Deng, C. X. 2014; 9 (1): e85749

  Abstract

  Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5-15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×10(4) at day 1 to 0.9±0.2×10(4) at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.

  View details for DOI 10.1371/journal.pone.0085749

  View details for PubMedID 24465680

  View details for PubMedCentralID PMC3899074

• Cell-based approaches to the engineering of vascularized bone tissue. Cytotherapy Rao, R. R., Stegemann, J. P. 2013; 15 (11): 1309-22

  Abstract

  This review summarizes recent efforts to create vascularized bone tissue in vitro and in vivo through the use of cell-based therapy approaches. The treatment of large and recalcitrant bone wounds is a serious clinical problem, and in the United States approximately 10% of all fractures are complicated by delayed union or non-union. Treatment approaches with the use of growth factor and gene delivery have shown some promise, but results are variable and clinical complications have arisen. Cell-based therapies offer the potential to recapitulate key components of the bone-healing cascade, which involves concomitant regeneration of vasculature and new bone tissue. For this reason, osteogenic and vasculogenic cell types have been combined in co-cultures to capitalize on the function of each cell type and to promote heterotypic interactions. Experiments in both two-dimensional and three-dimensional systems have provided insight into the mechanisms by which osteogenic and vasculogenic cells interact to form vascularized bone, and these approaches have been translated to ectopic and orthotopic models in small-animal studies. The knowledge generated by these studies will inform and facilitate the next generation of pre-clinical studies, which are needed to move cell-based orthopaedic repair strategies into the clinic. The science and application of cytotherapy for repair of large and ischemic bone defects is developing rapidly and promises to provide new treatment methods for these challenging clinical problems.

  View details for DOI 10.1016/j.jcyt.2013.06.005

  View details for PubMedID 23999157

  View details for PubMedCentralID PMC3832136

• Use of micro-computed tomography to nondestructively characterize biomineral coatings on solid freeform fabricated poly (L-lactic acid) and poly ((ε-caprolactone) scaffolds in vitro and in vivo. Tissue engineering. Part C, Methods Saito, E., Suarez-Gonzalez, D., Rao, R. R., Stegemann, J. P., Murphy, W. L., Hollister, S. J. 2013; 19 (7): 507-17

  Abstract

  Biomineral coatings have been extensively used to enhance the osteoconductivity of polymeric scaffolds. Numerous porous scaffolds have previously been coated with a bone-like apatite mineral through incubation in simulated body fluid (SBF). However, characterization of the mineral layer formed on scaffolds, including the amount of mineral within the scaffolds, often requires destructive methods. We have developed a method using micro-computed tomography (μ-CT) scanning to nondestructively quantify the amount of mineral in vitro and in vivo on biodegradable scaffolds made of poly (L-lactic acid) (PLLA) and poly (ε-caprolactone) (PCL). PLLA and PCL scaffolds were fabricated using an indirect solid freeform fabrication (SFF) technique to achieve orthogonally interconnected pore architectures. Biomineral coatings were formed on the fabricated PLLA and PCL scaffolds after incubation in modified SBF (mSBF). Scanning electron microscopy and X-ray diffraction confirmed the formation of an apatite-like mineral. The scaffolds were implanted into mouse ectopic sites for 3 and 10 weeks. The presence of a biomineral coating within the porous scaffolds was confirmed through plastic embedding and μ-CT techniques. Tissue mineral content (TMC) and volume of mineral on the scaffold surfaces detected by μ-CT had a strong correlation with the amount of calcium measured by the orthocresolphthalein complex-one (OCPC) method before and after implantation. There was a strong correlation between OCPC pre- and postimplantation and μ-CT measured TMC (R(2)=0.96 preimplant; R(2)=0.90 postimplant) and mineral volume (R(2)=0.96 preimplant; R(2)=0.89 postimplant). The μ-CT technique showed increases in mineral following implantation, suggesting that μ-CT can be used to nondestructively determine the amount of calcium on coated scaffolds.

  View details for DOI 10.1089/ten.TEC.2012.0495

  View details for PubMedID 23134479

  View details for PubMedCentralID PMC3662384

• Winner for outstanding research in the Ph.D. category for the 2013 Society for Biomaterials meeting and exposition, April 10-13, 2013, Boston, Massachusetts: Osteogenic differentiation of adipose-derived and marrow-derived mesenchymal stem cells in modular protein/ceramic microbeads. Journal of biomedical materials research. Part A Rao, R. R., Peterson, A. W., Stegemann, J. P. 2013; 101 (6): 1531-8

  Abstract

  Modular tissue engineering applies biomaterials-based approaches to create discrete cell-seeded microenvironments, which can be further assembled into larger constructs for the repair of injured tissues. In the current study, we embedded human bone marrow-derived mesenchymal stem cells (MSC) and human adipose-derived stem cells (ASC) in collagen/fibrin (COL/FIB) and collagen/fibrin/hydroxyapatite (COL/FIB/HA) microbeads, and evaluated their suitability for bone tissue engineering applications. Microbeads were fabricated using a water-in-oil emulsification process, resulting in an average microbead diameter of approximately 130 ± 25 μm. Microbeads supported both cell viability and cell spreading of MSC and ASC over 7 days in culture. The embedded cells also began to remodel and compact the microbead matrix as demonstrated by confocal reflectance microscopy imaging. After two weeks of culture in media containing osteogenic supplements, both MSC and ASC deposited calcium mineral in COL/FIB microbeads, but not in COL/FIB/HA microbeads. There were no significant differences between MSC and ASC in any of the assays examined, suggesting that either cell type may be an appropriate cell source for orthopedic applications. This study has implications in the creation of defined microenvironments for bone repair, and in developing a modular approach for delivery of pre-differentiated cells.

  View details for DOI 10.1002/jbm.a.34611

  View details for PubMedID 23554144

  View details for PubMedCentralID PMC4003500

• Assembly of discrete collagen-chitosan microenvironments into multiphase tissue constructs. Advanced healthcare materials Caldwell, D. J., Rao, R. R., Stegemann, J. P. 2013; 2 (5): 673-7

  Abstract

  Modular assembly of protein-polysaccharide microenvironments into 3D macroscale tissue constructs is reported. Rapid and simple centrifugation and vacuum molding methods are used to create cohesive multiphase constructs with prescribed geometries. Human fibroblasts are shown to survive in the microenvironments and in the macroscale constructs. Control of the spatial organization in engineered tissues is a key to recreating the complex tissue architectures needed for regenerative therapies.

  View details for DOI 10.1002/adhm.201200346

  View details for PubMedID 23184758

  View details for PubMedCentralID PMC4001864

• Delivery of mesenchymal stem cells in chitosan/collagen microbeads for orthopedic tissue repair. Cells, tissues, organs Wang, L., Rao, R. R., Stegemann, J. P. 2013; 197 (5): 333-43

  Abstract

  Microencapsulation and delivery of stem cells in biomaterials is a promising approach to repairing damaged tissue in a minimally invasive manner. An appropriate biomaterial niche can protect the embedded cells from the challenging environment in the host tissue, while also directing stem cell differentiation toward the desired lineage. In this study, adult human mesenchymal stem cells (MSC) were embedded in hydrogel microbeads consisting of chitosan and type I collagen using an emulsification process. Glyoxal and β-glycerophosphate were used as chemical and physical crosslinkers to initiate copolymerization of the matrix materials. The average size and size distribution of the microbeads could be varied by controlling the emulsification conditions. Spheroidal microbeads ranging in diameter from 82 ± 19 to 290 ± 78 µm were produced. Viability staining showed that MSC survived the encapsulation process (>90% viability) and spread inside the matrix over a period of 9 days in culture. Induced osteogenic differentiation using medium supplements showed that MSC increased gene expression of osterix and osteocalcin over time in culture, and also deposited calcium mineral. Bone sialoprotein and type I collagen gene expression were not affected. Delivery of microbeads through standard needles at practically relevant flow rates did not adversely affect cell viability, and microbeads could also be easily molded into prescribed geometries for delivery. Such protein-based microbeads may have utility in orthopedic tissue regeneration by allowing minimally invasive delivery of progenitor cells in microenvironments that are both protective and instructive.

  View details for DOI 10.1159/000348359

  View details for PubMedID 23571151

  View details for PubMedCentralID PMC3711684

• Noninvasive, quantitative, spatiotemporal characterization of mineralization in three-dimensional collagen hydrogels using high-resolution spectral ultrasound imaging. Tissue engineering. Part C, Methods Gudur, M., Rao, R. R., Hsiao, Y. S., Peterson, A. W., Deng, C. X., Stegemann, J. P. 2012; 18 (12): 935-46

  Abstract

  As tissue engineering products move toward the clinic, nondestructive methods to monitor their development and ensure quality are needed. In this study, high-resolution spectral ultrasound imaging (SUSI) was used to noninvasively characterize mineral content in collagen hydrogels. SUSI was used to generate three-dimensional (3D) grayscale (GS) images of construct morphology with submillimeter resolution. Spectral analysis of the backscattered radio frequency (RF) ultrasound signals was used to determine the midband fit (MBF) and slope of the linearized RF spectrum. These parameters are operator and instrument independent, and were used to characterize the spatial distribution of mineral in constructs supplemented with hydroxyapatite particles. GS and MBF correlated closely with mineral content, while slope was not dependent on concentration. SUSI also was used to monitor mineralization of collagen constructs by immersion in simulated body fluid (SBF) over 21 days. The construct surface was mineralized before the interior, and there was a dose-dependent effect of SBF concentration on degree of mineralization and deposited particle size. MBF density was closely correlated with the amount of calcium deposited. These data demonstrate that SUSI has utility as a noninvasive imaging method for quantitative analysis of mineralization in 3D protein constructs. Such techniques may assist the development of engineered orthopedic tissues.

  View details for DOI 10.1089/ten.TEC.2012.0180

  View details for PubMedID 22624791

  View details for PubMedCentralID PMC3968959

• Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials. Angiogenesis Rao, R. R., Peterson, A. W., Ceccarelli, J., Putnam, A. J., Stegemann, J. P. 2012; 15 (2): 253-64

  Abstract

  Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7 days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7 days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.

  View details for DOI 10.1007/s10456-012-9257-1

  View details for PubMedID 22382584

  View details for PubMedCentralID PMC3756314

• Exogenous mineralization of cell-seeded and unseeded collagen-chitosan hydrogels using modified culture medium. Acta biomaterialia Rao, R. R., Jiao, A., Kohn, D. H., Stegemann, J. P. 2012; 8 (4): 1560-5

  Abstract

  Induced biomineralization of materials has been employed as a strategy to increase integration with host tissue, and more recently as a method to control cell function in tissue engineering. However, mineralization is typically performed in the absence of cells, since hypertonic solutions that lack the nutrients and culture components required for the maintenance of cell viability are often used. In the present study, we exposed fibroblast-seeded three-dimensional collagen-chitosan hydrogels to a defined culture medium modified to have specific concentrations of ions involved in biomineralization. The modified medium caused a significant increase in calcium deposition in collagen-chitosan gels, relative to constructs incubated in a standard medium, though serum supplementation attenuated mineral deposition. Collagen-chitosan constructs became opaque over 3 days of mineralization in modified Dulbecco's modified Eagle medium (DMEM), in contrast to translucent control gels incubated in standard DMEM. Histological staining confirmed increased levels of mineral in the treated constructs. Rheological characterization showed that both the storage and loss moduli increased significantly in mineralized materials. Mineralization of fibroblast-seeded constructs resulted in decreased cell viability and proliferation rate over 3 days of incubation in modified medium, but the cell population remained over 75% viable and regained its proliferative potential after rescue in standard culture medium. The ability to mineralize protein matrices in the presence of cells could be useful in creating mechanically stable tissue constructs, as well as to study the effects of the tissue microenvironment on cell function.

  View details for DOI 10.1016/j.actbio.2012.01.001

  View details for PubMedID 22266029

  View details for PubMedCentralID PMC3289724

• Biomineralized composite substrates increase gene expression with nonviral delivery. Journal of biomedical materials research. Part A Rao, R. R., He, J., Leach, J. K. 2010; 94 (2): 344-54

  Abstract

  Current strategies to enhance gene transfer have focused on the development of vectors to increase the efficiency of DNA delivery. However, the extracellular matrix and microenvironment have a profound impact on numerous cellular activities including spreading and proliferation; two processes that have been associated with gene transfer efficiency. This study was designed to test the hypothesis that the presence of a biomineralized coating on biodegradable substrates would affect transgene expression following nonviral gene delivery. Thin films were prepared from polymeric microspheres, while biomineralized films were fabricated from microspheres previously soaked in modified simulated body fluid. Mineralized films were significantly more rigid and had widespread mineral coverage compared with nonmineralized substrates. Human mesenchymal stem cells (MSCs) were cultured on biomineralized or nonmineralized films and transfected with plasmid DNA condensed with linear polyethyleneimine (PEI). Compared with cells transfected on nonmineralized films, increases in gene expression were detected in the presence of biomineral at all charge ratios examined. We observed increased uptake of both PEI and DNA by cells on mineralized films. The results of these studies offer an approach to modulate gene delivery and improve the potential benefit of nonviral gene delivery approaches.

  View details for DOI 10.1002/jbm.a.32690

  View details for PubMedID 20186740

• Betacellulin inhibits osteogenic differentiation and stimulates proliferation through HIF-1alpha. Cell and tissue research Genetos, D. C., Rao, R. R., Vidal, M. A. 2010; 340 (1): 81-9

  Abstract

  Cellular signaling via epidermal growth factor (EGF) and EGF-like ligands can determine cell fate and behavior. Osteoblasts, which are responsible for forming and mineralizing osteoid, express EGF receptors and alter rates of proliferation and differentiation in response to EGF receptor activation. Transgenic mice over-expressing the EGF-like ligand betacellulin (BTC) exhibit increased cortical bone deposition; however, because the transgene is ubiquitously expressed in these mice, the identity of cells affected by BTC and responsible for increased cortical bone thickness remains unknown. We have therefore examined the influence of BTC upon mesenchymal stem cell (MSC) and pre-osteoblast differentiation and proliferation. BTC decreases the expression of osteogenic markers in both MSCs and pre-osteoblasts; interestingly, increases in proliferation require hypoxia-inducible factor-alpha (HIF-alpha), as an HIF antagonist prevents BTC-driven proliferation. Both MSCs and pre-osteoblasts express EGF receptors ErbB1, ErbB2, and ErbB3, with no change in expression under osteogenic differentiation. These are the first data that demonstrate an influence of BTC upon MSCs and the first to implicate HIF-alpha in BTC-mediated proliferation.

  View details for DOI 10.1007/s00441-010-0929-0

  View details for PubMedID 20165885

  View details for PubMedCentralID PMC2847694

• Biomimetic scaffolds fabricated from apatite-coated polymer microspheres. Journal of biomedical materials research. Part A Davis, H. E., Rao, R. R., He, J., Leach, J. K. 2009; 90 (4): 1021-31

  Abstract

  The deposition of a bonelike mineral on the surface of polymer scaffolds results in the formation of hybrid biomaterials, possessing enhanced osteoconductivity while retaining appropriate biodegradability. However, current methods of fabricating such composite scaffolds use a prolonged incubation process, which permits scaffold deformation and premature loss of incorporated macromolecules. We hypothesized that the fabrication of biomineralized polymer scaffolds could be achieved using premineralized polymer microspheres generated through incubation in a modified simulated body fluid (mSBF). We explored the material characteristics of these substrates and characterized the in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) when cultured on these novel scaffolds. Unlike scaffolds prepared using the conventional approach, premineralized scaffolds maintained their initial conformation after fabrication, achieved improved mineral distribution throughout the substrate, and enabled significantly greater incorporation efficiency of a model protein. We did not detect differences in osteogenic differentiation as determined by alkaline phosphatase activity and osteopontin secretion. However, we did observe a significant increase in cell-secreted calcium by hMSCs seeded on scaffolds prepared from premineralized polymer. These results demonstrate that the use of premineralized polymeric materials to fabricate biodegradable polymer scaffolds is an improved method for composite scaffold formation and may have numerous advantages for use in bone tissue engineering.

  View details for DOI 10.1002/jbm.a.32169

  View details for PubMedID 18655148