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Dr. Loay Al-Zube Scientific Contributions: Measuring Mechanical Tissue Properties of Pith-filled Plant Stems

alz loai — Sun, 17 May 2020 16:46:38 +0000

Plan Language Summary of Work

This research centered on the development of testing protocols that provide accurate and reliable measurements of the mechanical tissue properties of maize stalks. Our study presented for the first time 2 methods for measuring the mechanical tissue properties of maize stalks under compressive loading. Two different compressive modulus values were obtained: E-overall and E-local. The mean and standard deviation values for E-overall and E-local were (10.1 ± 1.5 and 12.8 ± 1.5 GPa, respectively). These test methodologies did not require that end faces were strictly parallel, and both methods produced consistent results (mean repeatability of 4%). Each of these methods possesses unique advantages and disadvantages. The overall compressive modulus technique provides a single, average value for all rind tissue in the specimen, but can be obtained relatively quickly. In contrast, the measurement of local modulus required multiple strain measurements, thus requiring additional tests, but provided results which are likely more accurate. although these measurements were developed and tested for dry maize specimens, the methods and principles introduced in this study are likely applicable for other types of plant stems, such as sorghum, reed, bamboo, etc.

We investigated further to measure the mechanical tissue properties of maize stalks under different loading modes (bending, compression and tensile), and to determine the accuracy and reliability of each test method. The three testing modes produced comparable elastic modulus values. All methods produced modulus of elasticity values that were within the range 6–16 GPa, indicating that each of these methods is effectively measuring the same physical feature of the maize rind, and that each method is relatively accurate. All methods exhibited good test-to-test repeatability values. Bending tests were found to be the fastest and most repeatable measurement approach. However, because the bending test requires a long slender specimen, it produces

an aggregate estimate of the modulus of elasticity. Compression tests require somewhat more sample preparation and testing time, but provide modulus of elasticity values that are more localized. Finally, the tensile testing approach is the most intensive in terms of sample

preparation, but produces the most localized measurement of the modulus of elasticity. This information can be used in creating structural models of maize stalk lodging, and to guide future experiments.

In a subsequent study, we provided for the first time a derivation of bending stress equations that takes into consideration many issues that are relevant to modeling bending stresses in plant stems. Six assumptions that are typically used in the derivation of bending stress equations used to predict mechanical stresses and failure of plant stems. We evaluated the derivation process in order to provide a clearer understanding of the accuracy and limitations of bending stress equations as applied to plant stems. This work involved using finite element models of plant stems to investigate and quantify the effect of each assumption. We derived bending stress equations that takes into consideration many issues that are relevant to modeling bending stresses in plant stems. Equations for stems with variation in cross-sectional stiffness were also developed in this work. We demonstrated that relatively few assumptions were required to obtain bending stress equations that are applicable to plant stems. We derived normal stresses, shear stresses, and combined stresses and presented their accompanying assumptions and notes regarding their applications. Our investigation should allow researchers to make well-informed decisions when modeling plant stems.

Resulting Publications

Loay Al-Zube, Wenhuan Sun, Daniel Robertson, and Douglas Cook. The Elastic Modulus of Maize Stems. Plant Methods. 2018 February; 14:11 (DOI: 10.1186/s13007-018-0279-6)
Loay Al-Zube, Daniel Robertson, Jean Edwards, Wenhuan Sun, and Douglas Cook. Measuring the Compressive Modulus of Elasticity of Pith-filled Plant Stems. Plant Methods. 2017 November; 13:99 (DOI: 10.1186/s13007-017-0250-y).
J. Stubbs, Navajit. S. Baban, Daniel J. Robertson, Loay Alzube and Douglas Cook. Bending Stress in Plant Stems: Models and Assumptions. In A. Geitmann and J. Grill, editors. Plant Biomechanics: From Structure to Function at Multiple Scales. 2018 Springer (ISBN 978-3-319-79099-2).

Technical Summary of Work

This work involved developing a robust testing protocol that provides accurate and reliable measurements of the compressive modulus of elasticity of the rind of pith-filled plant stems. Using compressional testing of dry, non-diseased maize stalk segments consisting of 2 nodes and the intervening internode tested between 2 self-aligning compression plates, we developed 2 methods for measuring the compressional modulus of elasticity of pith-filled node–node specimens. Two different compressive modulus values were obtained for each specimen in this study: E-overall and E-local. The mean and standard deviation values for E-overall and E-local were (10.1 ± 1.5 and 12.8 ± 1.5 GPa, respectively). Both methods had an average repeatability of ± 4%. The effect of sample position within the test fixture was quantified. We demonstrated that sample placement within ± 2 mm of the platen center had no significant effect on compressive modulus measurements made using our developed methods. The contribution of pith tissue to overall stiffness was assessed and was found to be approximately 4%. The two elastic modulus values were calculated using two different strain measurements. The overall compressive modulus technique provides a single, average value for all rind tissue in the specimen, but can be obtained relatively quickly. In contrast, the measurement of local modulus required multiple strain measurements, thus requiring additional tests, but provided results which are likely more accurate.

We also measured the modulus of elasticity of dry, mature maize rind tissues using different loading modes (bending, compression and tensile), and determined the accuracy and reliability of each test method. Our results demonstrated comparable elastic modulus values produced from the 3 testing modes ranged between 6 and 16 GPa. All three testing modes exhibited relatively favorable repeatability (i.e. test-to-test variation of < 5%). Five different compressive modulus values were obtained: E-Bending, E-Compression-overall, E-Compression-local, E-Tensile and E-Tensile-caliper. The mean and standard deviation values were 10.06 ± 1.51 GPa, 10.15 ± 1.47 GPa, 12.87 ± 1.56 GPa, 12.35 ± 1.51 GPa and 11.54 ± 1.37 GPa, respectively. Modulus values of internodal specimens were significantly higher than specimens consisting of both nodal and internodal tissues, indicating spatial variation in the modulus of elasticity between the nodal and internodal regions. Although these measurements were developed and tested for dry maize specimens, the methods and principles introduced in this study are likely applicable for other types of plant stems, such as sorghum, reed, bamboo, etc.

We analyzed, in a subsequent study and for the first time, six assumptions that are typically used in the derivation of bending stress equations used to predict mechanical stresses and failure of plant stems; orthotropic linear elasticity, normal stresses in z-direction are much larger than the normal stresses in x and y directions, bending is locally approximated by an arc, homogenous cross-sectional stiffness, shear stress (τ-yz) is much larger than shear stresses (τ-xz) & (τ-xy), and shear stress is a function of y only. We evaluated the derivation process in order to provide a clearer understanding of the accuracy and limitations of bending stress equations as applied to plant stems. This work involved using finite element models of plant stems to investigate and quantify the effect of each assumption. We derived bending stress equations that takes into consideration many issues that are relevant to modeling bending stresses in plant stems. Equations for stems with variation in cross-sectional stiffness were also developed in this work. We demonstrated that relatively few assumptions were required to obtain bending stress equations that are applicable to plant stems. We derived normal stresses, shear stresses, and combined stresses and presented their accompanying assumptions and notes regarding their applications. Our investigation should allow researchers to make well-informed decisions when modeling plant stems.

Summary of the Significant of Work

Corn (Zea mays L.) is the leading grain crop globally. In the U.S. alone, more than 250 billion tons are harvested annually, generating twice as much revenue as any other crop. Corn is deeply rooted in the global economy, being used in over 42,000 different applications, including the production of consumer goods, foods, pharmaceuticals, livestock feed, and fuel. As new uses and applications are discovered each year, the demand for corn continues to rise. However, 10-20% of this valuable crop is lost every year due to late season crop failure (referred to as stalk lodging). These losses significantly reduce productivity of farms, negatively impact individual farmers, and affect society at large by creating instability in the overall crop supply which directly affects the cost of consumer goods, commodities trading, fuel, food, and the broader economy. Reasons for crop failure have been challenging for plant geneticists to identify, measure, and control, largely because most crop research focuses on genetic, biological, and agronomic factors, not structural biomechanics. Over the past century, this focus on genetics has dramatically increased the economic importance of corn, increasing yield by over 400%. However, current high-yielding varieties of corn are very susceptible to stalk lodging. This is because yield and stalk size are often related. An increase in stalk size (i.e., a larger diameter) often causes a reduction in yield since the plant is utilizing energy to build a large stalk rather than grain. The solution to this problem lies in our ability to “design” a stalk that provides optimal strength, with minimal biological expense. Thus, our research is focused on identifying, quantifying and ranking key geometric and material features of corn stalk that are highly related to stalk strength but whose modification would only require minimal biological expense.

The many uses of corn, (biofuel, food products, pharmaceuticals, etc.), have triggered a drastically increased demand for corn production. However, yield increases in corn are currently limited by mechanical features and behavior which are not understood by plant scientists. The degree to which our society depends on corn is truly difficult to conceive. It feeds our livestock, fuels our cars and its byproducts are consumed by millions of Americans every day. In addition, its role as renewable resource has led to corn being used in an ever-increasing number of products, thereby increasing demand for worldwide corn production. Of particular importance is corn based ethanol production, which has increased by 3200% since 1982. Because of its ubiquitous use, even marginal improvements to the corn plant have the potential to result in billions of dollars of benefit to society. Stronger stalks will not only offset losses due to drought, disease and climate change, but will support the increased use of corn as a source of renewable energy. The merging of agronomy and biomechanics fields will lead to many scientific advances. While it is impossible to predict exactly what such advances will be it is likely that future collaborative efforts will result in stronger crops, fruit trees, and timber as well as an increased understanding of how genetic and environmental factors interact to determine the structural response of biological tissue.

In 2003, the Energy Biosciences Program (US Dept. of Energy) sponsored a workshop on Plant Systems Biology. Twenty top academic researchers discussed pressing grand challenges such as potential shortages in food and energy production. The primary recommendation of this workshop was a call for closer interactions between biologists and engineers toward the development of in silico (computational) models of plant physiology. Computational plant models provide numerous benefits, including (1) quantitative understanding of physilogical mechanisms; (2) understanding of interplay between various aspects of development; and (3) identifying areas of ignorance and guiding future research. The development of biomechanical models of crops has transformative potential, and is highly aligned with the recommendations and aspirations of top experts. Many important problems in plant physiology (form/function relationships, mechanical adaptation, etc.) can be solved only through the application of biomechanical principles. This project also has translational potential, as evidenced by our close collaborations with Monsanto scientists, which will insure the immediate application of research findings. The study of plant biomechanics provides an exceptional opportunity to obtain data that is appropriate for addressing many questions in human biomechanics. Like humans, plants exhibit wide variations in tissue properties and geometry. However, unlike humans both the genetic makeup and external environment can be directly controlled and manipulated in plants. In addition, plant tissues possess slower decay rates and are drastically reduced in cost as compared to human or animal studies. Thus, they provide an ideal opportunity to obtain extensive, correlated data using a biological system exhibiting many of the same features of human biomechanics but that is drastically reduced in cost, and allows more direct control over experimental variables. Data from our research will serve as a powerful and valuable resource for investigating the effects of biological variability on biomechanical models. This is a matter of pressing importance as data currently used in the creation of biomedical/biomechanical models and simulations is almost always uncorrelated.

Overall, our study promotes the use engineering principles, methodologies, and technologies to promote a closer interactions between biologists and engineers toward the development of in silico (computational) models of plant physiology.

Summary of Implementation/Influence of the Work

My recently published 2 papers and 1 book chapter related to this project have been cited, with a total of 29 citations. For specific citation details, please refer to my google scholar profile. A few notable citations include:

J. Stubbs, W. Sun, D.D. Cook. 2018. “Measuring the Transverse Young’s Modulus of Maize Rind and Pith Tissues”. Journal of Biomechanics. 2018 December; (https://doi.org/10.1016/j.jbiomech.2018.12.028). – In this study, the researchers developed a method for measuring the transverse Young’s modulus of maize stalk rind and pith tissues. Furthermore, X-ray computed tomography data was used to create a specimen-specific finite element model of the short, disc-shaped maize stalk segments tested. The specimen-specific finite element models provided estimates of the stress states in the stem under transverse loading, and these stress states accurately predicted the location of failure in transverse test specimens. These researchers from New York University (New York, USA) and Brigham Young University (Utah, USA) cited 2 of my papers and specifically mentioned our studies in the context of developing alternative bending, compression and tensile methods for quantifying and reporting tissue properties of maize tissues.
Charrier, H. Rabille, B. Billoud. 2018. “Gazing at Cell Wall Expansion Under Golden Light”. Trends in Plant Science. 2018, November; (https://doi.org/10.1016/j.tplants.2018.10.013) – In this report the researchers represented how much of the functional relationship between cell wall chemistry and intrinsic mechanics on the one hand, and growth on the other hand, has been uncovered in brown algae. These researchers from CNRS-Sorbonne University (Roscoff, France) cited 1 of my papers and specifically mentioned our study in the context of quantifying and reporting bending, compression and tensile Young’s Modulus of maize stems.

The post Dr. Loay Al-Zube Scientific Contributions: Measuring Mechanical Tissue Properties of Pith-filled Plant Stems appeared first on ALZUBE.com.

Dr. Loay Al-Zube Scientific Contributions: The Effects of local Recombinant Human Bone Morphogenic Protein-2 (rhBMP-2) delivery and Mesenchymal Stem Cells (MSC) augmentation on Bone Allograft Incorporation in the presence of Diabetes Mellitus

alz loai — Sun, 17 May 2020 16:31:02 +0000

Plain Language Summary of Work

This research centered on the investigation of the effects of Diabetes Mellitus (DM), local delivery of an exogenic genetic-protein (rhBMP-2), and stem cell augmentation on bone segmental defects healing in the presence of diabetes mellitus in a rat model. Our study is the first to analyze the effects of DM in a segmental defect model and treated with the application of a local rhBMP-2 delivery system. We demonstrated that animals treated with rhBMP-2/collagen carrier–treated had significantly increased bridging of the defects and increased radiographic scores and increased histomorphometric and mechanical testing parameters of new bone formation compared with the buffer/collagen-treated group in both DM and non-DM rats. This study demonstrated the effectiveness of rhBMP in the presence of DM in a segmental defect and provides insight in the potential role of rhBMP-2 in this difficult clinical application.

We investigated further to analyze the effect of MSC augmentation upon bone Allograft (Allo) incorporation, using histomorphometric (histological) outcome parameters upon a segmental defect model in the presence of diabetes mellitus (DM). Our study demonstrated that DM delayed Allo incorporation and bone formation in DM rats compared to that of its non-DM counterpart. Our results support the concept that MSC augmentation significantly increases bone formation in allograft incorporation in the presence of DM. The amount of bone increased greater than 300% from an average of (1.07 mm²) in DM/DBM animals to (3.57 mm²) in DM/MSC animals at the early time point of 4 weeks. Additionally, more mature bone was observed with local MSC augmentation in the DM animals at both 4 and 8 weeks. The current study confirms the potential of MSC augmentation upon allograft incorporation in a DM segmental defect model by demonstrating that a DM segmental defect treated with MSC is histomorphometrically indistinguishable from a non-DM segmental defect utilizing a current mainstay treatment of DBM alone. Our study is the first study exploring MSC as a potential adjuvant for allograft incorporation in a DM population.

Resulting Publications

Publications (Journal articles):

Vikrant Azad, Eric Breitbart, Loay Al-Zube, Sloane Yeh, Ann Marie Simon, J. Patrick O’Connor, Sheldon Lin. “rhBMP-2 enhances the bone healing response in a diabetic rat segmental defect model”. J Orthop Trauma. 2009 Apr; 23(4):267-76.
Eric A. Breitbart, Sharonda Meade, Vikrant Azad, Sloane Yeh, Loay Al-Zube, Yee-Shuan Lee, Joseph Benevenia, Treena Livingston-Arinzeh, Sheldon Lin. “Mesenchymal Stem Cells Accelerate Bone Allograft Incorporation in the Presence of Diabetes Mellitus”. J Orthop Res. 2010 July; 28(7): 942-949.
Jacobsen K.; Szczepanowski K.; Al-Zube L. A., Kim J., and Lin S. S., 2007. “The Role of Intraoperative Bone Marrow Aspirate Stem Cell Concentration as a Bone Grafting Technique”. Techniques in Foot & Ankle Surgery. 2008 June; 7(2):84-89.

Technical Summary of Work

This work involved the investigation of the effects of local recombinant human bone morphogenic protein-2 (rhBMP-2) delivery on bone healing in segmental femoral defects in the presence of diabetes mellitus. Using collagen sponge soaked with rhBMP-2 inserted in a mid-diaphyseal 3.0-mm femoral defect in rats fixed with polyimide plate and stainless steel screws, we demonstrated the effectiveness of rhBMP in increasing new bone formation in a segmental defect in the presence of diabetes Mellitus. Radiographic, histomorphometric, neoangiogenesis, and mechanical testing were employed. Radiographs assessed over a 6-point grading system showed statistically significant improvement in scores in rhBMP-2–treated rats at 6 weeks (P < 0.001). Histomorphometric analysis showed statistically significant increase in area of new bone formation between rats treated with rhBMP-2 at both 3 and 6 weeks (P < 0.001). On Platelet endothelial cell adhesion molecule-1 staining at 3 weeks, the mean number of vessels in rhBMP-2–treated DM rats was 12.76 ± 5.43/mm²compared with 4.49 ± 1.89/mm² in buffer (collagen sponge with saline) treated DM rats (P = 0.034). On mechanical testing, all 4 DM/buffer rats had nonunion. In DM/rhBMP-2 rats, the torque to failure and torsional rigidity values were 393.57±233.3 (P < 0.03) and 29,711± 6224 (P < 0.002), respectively. Further, our model also revealed that a single application of rhBMP-2 resulted in new bone formation in the DM group comparable to the non-DM group, thus demonstrating the ability of rhBMP-2 in negating the deleterious effects of DM on new bone formation.

We also explored the effects of Mesenchymal Stem Cells (MSC) augmentation upon bone allograft (Allo) incorporation in the presence of diabetes mellitus (DM). Using segmental (5 mm) femoral defects created in non-DM and DM rats and treated with allografts containing demineralized bone matrix (DBM) or DBM with MSC augmentation, we demonstrated that the presence of DM resulted in less mature bone 4 weeks after treating the defect with Allo DBM (P< 0.001). However, there was significantly more mature bone in the DM/MSC group when compared to the DM/DBM group at both 4 and 8 weeks (p<0.001 and p=0.004). Furthermore, significantly more bone formation was observed in the DM/MSC group compared to the DM/DBM group at the 4-week time point (p<0.001). The results of this study suggest that MSC are a potential adjunct for bone regeneration when implanted in an orthotopic site in the presence of DM. The primary strength of this study is that, to our knowledge, this is the first study exploring MSC as a potential adjuvant for allograft incorporation in a DM population. This study demonstrates the potential role of MSC as an adjunct, to address the clinical problems of delayed allograft incorporation in the presence of DM.

Summary of the Significance of the Work

Critically sized bone defects often occur as a result of metabolic diseases, trauma, or cancer. Bone lesions above a critical size become atrophic and scarred rather than regenerate, leading to nonunion, and may require revision surgery to restore lost function. More than one million orthopedic operations are performed in the United States each year for reconstructive surgery,

trauma, or abnormal skeletal defects. Often, large amounts of autologous, or alternatively, large bulk allograft (Allo) are needed in the surgical procedure to achieve the reconstructive goals. Although the current technique for bone grafting is autologous cancellous bone, various problems are associated with its acquisition, including donor site morbidity, loss of function, and a limited supply. Another major issue affecting both autograft and Allo incorporation is the presence of systemic diseases like diabetes mellitus (DM). Currently, 21 million Americans are diagnosed with DM, with over 600,000 new cases diagnosed each year. Despite the paucity of literature on the impact of DM on bone formation and fracture healing, the few clinical series demonstrate a significant deleterious effect of this systemic disease. DM patients experience a significant delay to union, uniting at an average of approximately two times longer than fractures in patients without DM. Our research is especially valuable because we demonstrated that rhBMP-2 can be utilized to significantly enhance bone healing response in diabetic segmental defect model. We also demonstrated that Mesenchymal Stem Cells (MSC) augmentation accelerate bone allograft incorporation in the presence of diabetes mellitus. Overall, our study promotes the use of rhBMP-2 and MSC augmentation in bone regeneration and formation and strongly support continued research in the use of osteogenic markers, non-osseous carriers for MSC to decrease the amount of bone needed for reconstructive surgery. Additional studies may focus on application of temporal control of genetic protein release for the determination of which specific phase of bone remodeling is up-regulated.

Summary of the Implementation/Influence of the Work

My 3 papers related to this project have been widely cited, with a total of 99 citations. For specific citation details, please refer to my google scholar profile. A few notable citations include:

Yu, W. Zhou, Y. Song et al. 2011. “Development of mesenchymal stem cell-implant complexes by cultured cells sheet enhances osseointegration in type 2 diabetic rat model”. Bone. 2011 September; 49(3): 387-394. – In this study, the researchers developed a mesenchymal stem cells (MSC)-implant complex as a novel tissue engineering approach that promotes osseous healing in the presence of diabetes mellitus type 2. These researchers from the Fourth Military Medical University (Xi’an, PR China) cited one of my papers and specifically mentioned our study in the context of demonstrating that their findings were similar to ours and support that MSCs may accelerate bone healing in the DM rat model.
K. Udehiya, Amarpal, H.P. Aithal et al. 2013. “Comparison of autogenic and allogenic bone marrow derived mesenchymal stem cells for repair of segmental bone defects in rabbits”. Research in Veterinary Science. June 2013; 94(3): 743-752. – In this study, the researchers compared the use of Autogenic and Allogenic bone marrowderived mesenchymal stem cells (BM-MSCs) for repair of bone gap defect in rabbits. These researchers from the Indian Veterinary Research Institute (Izatnagar, India) cited one of my papers and specifically mentioned our study in the context of demonstrating that their findings were similar to ours, specifically observing more bony union in mesenchymal stem cells treated group as compared to the groups treated by demineralized bone matrix (DBM) in diabetic animals at the 4 week time point, and that the newly formed bone showed early initiation of remodeling in treatment groups.
I. Ko, L.S. Coimbra, C. Tian et al. 2015. “Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism”. Diabetologia. March 2015; 58(3): 633-642. – This work was supported by National Institute of Health/National Institute of Arthritis and Muscular and Skin Diseases (NIH/NIAMS) grant. Inthis study, the researchers demonstrated that Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. The researchers suggested that reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximize regenerative potential in diabetic patients. These researchers from University of Pennsylvania (Philadelphia, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating that the treatment of diabetic fractures with MSCs can promote healing.
M. Wojtowicz, A. Shekaran, M.E. Oest et al. 2010. “Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair”. Biomaterials.March 2010; 31(9): 2574-2582. – This work was supported by the National Institutes of Health and Georgia Tech/Emory National Science Foundation ERC on the Engineering of Living Tissues. Inthis study, the collagen-mimetic peptide, GFOGER, was used by the researchers to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. The researchers demonstrated a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. These researchers from Georgia Institute of Technology (Atlanta, GA, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating that although the GFOGER sequence is present in native full-length collagen, collagen alone does not enhance bone formation in critically-sized defects.
Shekaran, J. Garcia, A. Clark et al. 2014. “Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as BMP-2 delivery vehicle”. Biomaterials.July 2014; 35(21): 5453-5461. – This work was funded by the National Institutes of Health, USA and by the Singaporean Agency for Science, Technology and Research. In this study, the researchers engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. The findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential. These researchers from Georgia Institute of Technology (Atlanta, GA, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating the differences of healing induced by the GFOGER-tethered PEG gels compared to collagen foams.
S.R. Angle, K. Sena, D.R. Sumner et al. 2012. “Healing of rat femoral segmental defect with bone morphogenetic protein-2: A dose response study”. Journal of Musculoskelet Neuronal Interact. 2012; 12(1): 28-37. – This work was funded in parts by the National Institutes of Health – National Institute of Arthritis and Musculoskeletal and Skin Diseases, and a Medtronic grant from the Orthopaedic Trauma Association. In this study, the researchers used a segmental defect model to assess the dose response of rhBMP-2 using quantitative and qualitative endpoints. The researchers demonstrated that all doses of rhBMP-2 result in new bone formation. However, there is an optimum dose of 12 μg of rhBMP-2 for bone repair in the model used, above which and below which less stimulation of bone occurs. These researchers from Rush University Medical Center (Chicago, IL, USA) and University of Illinois (Chicago, IL, USA) cited one of my papers and specifically mentioned our study in the context of demonstrating that Recombinant human BMP-2 (rhBMP-2) has been most successful for bone repair in animal models.

The post Dr. Loay Al-Zube Scientific Contributions: The Effects of local Recombinant Human Bone Morphogenic Protein-2 (rhBMP-2) delivery and Mesenchymal Stem Cells (MSC) augmentation on Bone Allograft Incorporation in the presence of Diabetes Mellitus appeared first on ALZUBE.com.

Dr. Loay Al-Zube Scientific Contributions: The effects of local delivery of Insulin on osseous repair

alz loai — Sun, 17 May 2020 14:53:22 +0000

Plain Language Summary of Work

This work tested whether local insulin therapy could accelerate femur fracture repair in normal, non-diabetic rats.Histomorphometry, radiographic scoring, and torsional mechanical testing were used to measure fracture healing. Using a closed mid-diaphyseal fracture created in the right femur in normal, non-diabetic rats treated with 2 doses of insulin (10 and 20 units) delivered in a calcium sulfate carrier, we demonstrated that low dose insulin (10 units) had a positive effect on biomechanical properties, supported with higher qualitative radiographic scoring, but did not appear to have a significant effect on any of the histological parameters measured. On the other hand, we demonstrated that the high dose (20 units) treatment had significantly increased the amount of cartilage in the fracture callus at 7- and 14-days post-fracture, consistent with enhanced chondrogenesis, but this did not result in better healing outcomes as determined by mechanical testing. Further, our study results also indicate that insulin dose or duration of insulin treatment are critical variables in controlling the fracture healing process since the high dose did not have a positive effect upon the biomechanical parameters at 4 weeks after fracture. The results of this study suggest that locally delivered insulin is a potential therapeutic agent for treating bone fractures. Further studies are necessary, such as large animal proof of concepts, prior to the clinical use of insulin for bone fracture treatment.

We investigated further to quantify the effects of local, early delivery of Ultralente insulin (UL) on various fracture healing parameters in non-diabetic BB Wistar rats. Quantification of insulin levels showed a rapid release of insulin from the fractured femora, demonstrating complete release at 2 days. We demonstrated that this release significantly enhanced mineralization-related markers (Col1a2, osteopontin) with UL insulin treatment 4- and 7- days post fracture. Our work also demonstrated significant differences in vascularity (75% increase in the insulin group) and vessels-forming cells between the treatment and control groups at day 7, with more evident in the insulin-treated group. These early changes resulted in a significant increase in percent-mineralized tissue in the UL-treated animals compared with controls 21 days post-fracture, and in a significantly greater mechanical strength 4 weeks post-fracture. Our study demonstrated that acute, local insulin treatment immediately after fracture promoted healing in non-diabetic rats.

Although local UL insulin results suggest a promising new therapeutic strategy for treating bone fractures and other skeletal injuries, additional studies are needed to understand how dose and duration of treatment affect bone regeneration as well as to characterize insulin’s mechanism of action. Faced with the challenge of finding possible delivery vehicles for insulin to enhance bone fracture healing, 2 calcium salts carriers, Calcium sulfate (CaSO4) and β-tricalcium phosphate (TCP), were investigated as possible delivery vehicles. Our findings showed that; in vitro, beta tricalcium phosphate sustained a prolonged insulin release compared to calcium sulfate. Also, our results showed the strong solidification tendency of calcium sulfate as compared to the paste-like state of beta tri-calcium phosphate carrier in a saline solution at 37°C temperature.

Resulting Publications

Publications (Ph.D. Dissertation):

Rutgers University (Formerly: University of Medicine and Dentistry of New Jersey). Newark, NJ, USA. Biomedical Engineering, April 2008 Ph.D. Dissertation: The Role of Local Growth Factor Delivery on BoneFracture Healing: Recombinant Human Platelet Derived Growth Factor and Insulin.

Publications (Journal articles):

Park AG, Paglia DN, Al-Zube L, Hreha J, Vaidya S, Breitbart E, Benevenia J,O’Connor JP, Lin SS. Local Insulin Therapy Enhances Fracture Healing in a Rat Model. Journal of Orthopaedic Research. 2013 May; 31(5): 776-782.
David N. Paglia, Aaron Wey, Eric A. Breitbart, Jonathan Faiwiszewski, Siddhant K. Mehta, Loay Al-Zube, Swaroopa Vaidya, Jessica A. Cottrell, Dana Graves, Joseph Benevenia, J. Patrick O’Connor, Sheldon S. Lin. Effects of Local Insulin Delivery on Subperiosteal Angiogenesis and Mineralized Tissue Formation during Fracture Healing. Journal of Orthopaedic Research. 2013 May; 31(5): 783-791.
Loay A. Al-Zu’be, Thakir D. Al-Momani, Osama M. Al-Bataineh, and Lubna H. In vitro Characterization of Calcium Salts as Delivery Vehicles for Insulin. Journal of Biomimetics Biomaterials and Tissue Engineering. 2013 June; 17: 53-58.

Publications (Patents):

Sheldon Suton Lin, Ankur Gandhi, James Patrick O’Connor, Loay A. Al-Zube, Joseph Benevenia, Russell Parsons. Localized insulin delivery for bone healing. US Patent no. US7763582, assigned to the University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.

Technical Summary of Work

This work tested whether local insulin therapy could accelerate femur fracture repair in normal, non-diabetic rats.Histomorphometry, radiographic scoring, and normalized torsional mechanical testing were used to measure fracture healing at multiple time points. Using a closed mid-diaphyseal fracture created in the right femur in normal, non-diabetic rats treated with 2 doses of insulin delivered in a calcium sulfate carrier, we demonstrated that low dose insulin (10 units) treatment resulted in significantly greater percent-torque to failure as compared to all other treatment groups (p < 0.05) and greater percent-shear stress as compared to the negative control groups (p < 0.05), supported with higher qualitative radiographic scoring, but did not appear to have a significant effect on any of the histological parameters measured. Also, we demonstrated that at 7- and 14-days post-fracture the high dose (20 units) treatment resulted in significant increase in percent-cartilage in the fracture callus as compared to the low dose treatment and negative control groups (p < 0.05), consistent with enhanced chondrogenesis, but this did not result in better healing outcomes as determined by mechanical testing. Further, we demonstrated that when local UL insulin was delivered with a calcium sulfate carrier, tissue insulin levels were significantly greater (p < 0.05) in the fractured femora than the unfractured/untreated femora of the rats treated with high dose within the first 4 days. No statistically significant difference in plasma insulin levels was detected. Although local UL insulin results suggest a promising new therapeutic strategy for treating bone fractures and other skeletal injuries, additional studies are needed to understand how dose and duration of treatment affect bone regeneration as well as to characterize insulin’s mechanism of action.

We also quantified the effects of local intramedullary delivery of Ultralente insulin (UL) on subperiosteal angiogenesis and mineralized tissue formation during fracture healing in a non-diabetic rat model. Histomorphometry, protein qualification, normalized torsional mechanical testing, gene expression analysis, and immunohistochemistry were used and quantified at multiple time points. Using a closed mid-diaphyseal fracture created in the right femur in normal, non-diabetic rats treated with either saline (control group) or UL insulin (experimental group) administrated in the intramedullary canal, we demonstrated that percent-mineralized tissue in insulin-treated animals was significantly greater than controls (p = 0.021) at day 21 post-fracture, however, this effect was not sustained at later time points. UL insulin treatment resulted in a 39% increase in the maximum torque to failure (p= 0.008) and a 58% increase in maximum shear stress (p=0.018) as compared to the control group 4 weeks post fracture.

Our study also demonstrated a significant increase in early osteogenic gene expression, but not chondrogenic gene expression, in animals treated with insulin. Specifically osteopontin mRNA level at 4 day following fracture (p=0.015) and Colla2 mRNA level at 7 days following fracture (p=0.001).

Further, our study revealed significant increase in the average blood vessel (BV) density (number of BVs/mm2 callus area) and in the number of VEGF-C+ cells within the subperiosteal region in the insulin-treated group compared with controls (p = 0.008 and p < 0.001, respectively). In this project differences in local insulin levels were detected between the right, fractured femora and left, intact femora of the rats treated with insulin within the first 12–48 h (significantly higher at 48 h [p = 0.045]), but were largely depleted by 96 h. Overall, our work demonstrated that acute, local insulin treatment immediately after fracture promoted healing in non-diabetic rats through an increase in osteogenic gene expression, subperiosteal angiogenesis, and mineralized tissue formation. While the current study did find significant increases in early osteogenic gene expression, no such changes were evident for chondrogenic gene expression. This finding is possibly due to the rapid release of insulin from the fractured femora.

In a subsequent study we investigated release kinetics of insulin from two different osteoconductive carriers, calcium sulfate (CaSO4) and β-tricalcium phosphate (TCP), in vitro at multiple time points. Our results demonstrated an early burst in insulin release when delivered via the calcium sulfate carrier and a continuous and rapid release within the 1st 12 hours, whereas, β-tricalcium phosphate carrier caused significantly slower release kinetics within the same period (p < 0.05). Further, our study demonstrated the solidification tendency of the calcium sulfate carrier as opposed to the paste-like state of the β-tricalcium phosphate carrier 48 hours after being placed in a saline solution at 37°C. Our solidification observation indicates that CaSO4 might serve as a stabilizing material around the fracture site due to its high solidification tendency; however, the solidification of CaSO4 might lead to microvasculature obstruction during revascularization in the healing process. Overall, calcium sulfate (CaSO4) caused the release of a large, early-burst of insulin immediately after its placement in the saline solution followed by a rapid release of its content of insulin within 12 hours, while beta tri-calcium phosphate (TCP) resulted in a smaller early-burst of insulin followed by a continuous and sustainable release projected linearly for up to ~12 hours.

Summary of the Significance of the Work

Over 10 million surgical procedures are performed annually in the United States to treat musculoskeletal injuries (Young CS et. al. 2009). Musculoskeletal conditions are among the most disabling and costly conditions suffered by Americans. Aggregate total Musculoskeletal medical care expenditures in the United States reached $796.3 billion in 2011, from which $190.6 billion are the annual cost of injuries and fractures (The burden of musculoskeletal diseases in the united states, 2014). Approximately 10% of the 7.9 million annual fracture patients in the United States experience nonunion and/or delayed unions, which have a substantial economic and quality of life impact (The burden of musculoskeletal diseases in the united states, 2014). Although many orthopaedic problems existed in the past now have practical solutions because of the collaborative basic and clinical research, impaired fracture healing represents an ongoing failure of initial fracture management (Hayda RA et. al. 1998). Therefore, any research that helps to mitigate health outcomes associated with fracture nonunion and/or delayed unions is very valuable to the US in particular. Our research is especially valuable because we demonstrated that local delivery of insulin, which is widely available and relatively cheap, can be utilized as a therapeutic agent to significantly promote fracture healing in health and compromised conditions such as diabetes and osteoporosis. We also demonstrated the dose dependent effect of local insulin delivery on early and late fracture healing stages. We also identified possible research avenues needed to understand how dose and duration of treatment affect bone regeneration as well as to characterize insulin’s mechanism of action. We also identified possible methods to deliver insulin via a carrier that would avoid burst dosing effects and early dissipation of insulin that will increase the effectiveness and safety. Overall, our studies promote the use of insulin to promote bone healing in animal models and strongly support continued research in the use of insulin. Insulin is much cheaper than many other pharmaceutical agents, so further verification of its clinical value is also relevant to the reduction of healthcare costs.

Summary of Implementation/Influence of the Work

My patent was licensed to CreOsso, LLC, Montclair, NJ, USA (http://www.creosso.com/index.html). CreOsso, LLC. seeks to leverage close to 10 years of research into the effects of local delivery of insulin and insulin-mimetics on bone regeneration in long bones, extremities and the spine. My 3 papers and 1 patent related to this project have been widely cited, with a total of 69 citations. For specific citation details, please refer to my google scholar profile. A few notable citations include:

Yuasa, N.A. Mignemi, J.V. Barnett et al. 2014. “The temporal and spatial development of vascularity in a healing displaced fracture”. Bone. 2014 October; 67:208-221. – In this study, the researchers combining novel techniques of bone angiography and a reproducible murine femur fracture model to demonstrate for the first time the complete temporal and spatial pattern of revascularization in a displaced/stabilized fracture. These researchers from Vanderbilt University Medical Center (Nashville, TN) cited 2 of my papers and specifically mentioned our studies in the context of demonstrating the significant of addressing vascular dysfunction in reducing fracture healing complications.
O. Malekzadeh, M. Ransjo et al. 2016. ‘Insulin released from titanium discs with insulin coatings – kinetics and biological activity’. Journal of Biomedical Materials Research Part B. 2016 May; 105(7):1847-1854. – These researchers ran an in vitro study where human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro‐chemiluminescence immunoassay, furthermore, they evaluated the biological effects of the released insulin on human osteoblast‐like MG‐63 cells. These researchers from the University of Gothenburg (Gothenburg, Sweden) cited 2 of my papers in the context of demonstrating the anabolic significance of insulin in bone healing and the different methods we used to locally administrate insulin around the fracture site via injection or in combination with carriers.
B. Clifton, D.N. Paglia et. al. 2014. ‘Effects of Wnt5a Haploinsufficiency on Bone Repair’. Journal of Orthopaedic Trauma’. 2014 August; 28(8); e191-e197. – These researchers, to better understand the effect of the Wnt5a on bone repair, carried out Femoral fracture experiments on Wnt5a+/+ and Wnt5a+/− mice. These researchers from the University of Connecticut Health Center (CT, USA) cited 2 of my papers in the context of demonstrating the testing methodologies used.
Zhu, Y. Zhu, B. Ni et. al. 2014. ‘Mesoporous Silica Nanoparticles/Hydroxyapatite Composite Coated Implants to Locally Inhibit Osteoclastic Activity’. 2014; 6(8); 5456-5466. – These researchers developed a composite coating drug delivery system, on the surface of stainless Kirschner wires, composed of HA and mesoporous silica nanoparticles (MSNs) to provide functions of drug delivery for zoledronic acid molecules to resist unwanted bone resorption and accelerate fracture healing. These researchers from the Second Military Medical University (Shanghai, P. R. China) cited one of my papers in the context of demonstrating that biological coated-internal fixation implants have shown great potentials in accelerating fracture healing through incorporating and locally release bioactive components.

The post Dr. Loay Al-Zube Scientific Contributions: The effects of local delivery of Insulin on osseous repair appeared first on ALZUBE.com.

Dr. Loay Al-Zube Scientific Contributions: The effects of local delivery of recombinant-human platelet-derived growth factor-BB (rhPDGF-BB) on osseous repair in the presence of diabetes mellitus

alz loai — Sun, 17 May 2020 06:08:56 +0000

Plain Language Summary of Work

This research centered on investigating the effects of the local application of a known growth factor, recombinant-human platelet-derived growth factor BB (rhPDGF-BB), on compromised bone healing in diabetic rats. Our study revealed for the first time that the local application of rhPDGF-BB around the fracture site increased the number of callus cells in diabetic rats, which increases the mechanical strength of bone 8 weeks post-fracture. We demonstrated for the first time in the literature that local rhPDGF-BB treatment significantly enhances bone fracture healing in the presence of diabetes mellitus. Furthermore, our findings indicate that local rhPDGF-BB treatment is highly targeted and therefore unlikely to have significant off-target effects (side effects).

We further investigated the histology of fractured femora 12 weeks post-fracture to identify the long-term effects of local rhPDGF on bone formation. If the rats were treated with the high dose rhPDGF-BB (75 µg) an increased bone formation was observed at the fracture site. This support the hypothesis that local treatment of rhPDGF-BB ultimately leads to more bone formation. Our findings showed a dose-dependent effect. As such, our study provides a blueprint for determining which dose of rhPDGF-BB should be used locally to enhance early and late stages of bone fracture healing in the presence of diabetes mellitus as well as identifying other possible applications for rhPDGF in bone metabolism and repair.

Resulting Publication

Ph.D. Dissertation:

Rutgers University (Formerly: University of Medicine and Dentistry of New Jersey). Newark, NJ, USA. Biomedical Engineering, April 2008 Ph.D. Dissertation: The Role of Local Growth Factor Delivery on BoneFracture Healing: Recombinant Human Platelet Derived Growth Factor and Insulin.

Journal Articles:

Al-Zube, Loay; Breitbart, Eric A.; O’Connor, J. Patrick; Parsons, J. Russell; Bradica, Gino; Hart, Charles E.; Lin, Sheldon S. Recombinant Human Platelet-Derived Growth Factor BB (rhPDGF-BB) and Beta-Tricalcium Phosphate/Collagen Matrix Enhance Fracture Healing in a Diabetic Rat Model. Journal of Orthopaedic Research. 2009 Aug;27(8):1074-81.

Technical Summary of Work

This work involved evaluating the dose-dependent effect of rhPDGF-BB delivered in a b-TCP/Type I bovine collagen matrix on fracture healing in the diabetic BB Wistar rat model. Low (22 µg) and high (75 µg) doses of recombinant human PDGF-BB (rhPDGF-BB) were applied directly to femur fracture sites in BB Wistar diabetic rats that were then compared to untreated or vehicle-treated animals. Using diabetic-prone BB Wistar rats that had become diabetic and that were treated with insulin implants to maintain blood glucose levels between 300 and 500 mg/dl, we demonstrated that fracture callus cell proliferation was significantly higher in the diabetic rats treated with rhPDGF-BB, indicating that rhPDGF-BB treatment can ameliorate the deficit in callus cell proliferation observed in diabetic animals. Torsional mechanical testing also indicated that low dose rhPDGF-BB treatment (22 µg) significantly increased fracture callus peak torque (p<0.05) at 8 weeks after healing as compared to controls, supporting the hypothesis that rhPDGF-BB induced fracture callus cell proliferation and led to a subsequent increase in the structural properties of the callus in the diabetic rat.

We also prepared and analyzed histological sections of fractured femora. While histomorphometric analysis at 12 weeks post-fracture showed no significant difference in callus area, a significant increase in percent mineralized area was observed in the calluses treated with high dose rhPDGF-BB as compared to low dose rhPDGF-BB or control groups. These data suggest that rhPDGF-BB treatment may have a long-term effect that enhances the later stages of fracture healing in diabetic animals. High dose rhPDGF-BB treatment significantly increased early callus cell proliferation but had no effect later on callus mechanical properties. In contrast, low dose rhPDGF-BB increased early callus cell proliferation and later on callus biomechanical properties. We interpret these data to indicate that in this model, high dose rhPDGF-BB treatment may delay differentiation of mesenchymal cells into chondrocytes and osteoblasts by extending the proliferative phase.

Summary of the Significance of the Work

Twenty million Americans have diabetes mellitus (diabetes) and 500,000 to 1,000,000 new cases are diagnosed each year. The prevalence of diabetes is estimated to exceed 30 million patients in the United States by 2010 (currently 30.2 million patients, National Diabetes Statistics Report, 2017). The association between diabetes and impaired osseous healing has been documented in clinical and experimental settings. In 2012, the US faced over $ 245 billion in direct and indirect medical costs as a result of diabetes (National Diabetes Statistics Report, 2017), and therefore any research that helps to mitigate health outcomes associated with diabetes is very valuable to the US in particular. Our research is especially valuable because we demonstrated that local delivery of growth factors (such as rhPDGF-BB), which had previously not been widely used as a therapeutic option due to technology/carrier limitations, can be utilized to significantly reverse compromised bone fracture healing in diabetes patients. We also demonstrated that local rhPDGF treatment has dose dependent effects on early and late stages of fracture healing in diabetic animals. Overall our study promotes the use of biological growth factors and proteins locally as a therapeutic option to reverse compromised fracture healing in diabetes patients, and strongly supports continued research in the use of rhPDGF-BB delivered locally via suitable possible drug-delivery systems to significantly enhance fracture healing and bone regeneration. Further research in developing and validating biomaterials as carriers for local, long-term, and sustainable release of rhPDGF-BB is relevant to further enhance bone fracture healing, which will help further reduce healthcare costs.

Summary of the Implementation/Influence of the Work

BioMimetic Therapeutics, Inc. (Franklin, TN) supported my research. Based on my pre-clinical research findings, BioMimetic Therapeutics conducted clinical trials. The clinical trials resulted in acquiring BioMimetic Therapeutics by Wright Medical. A clinical product named AUGMENT® BONE GRAFT was developed. Augment ® Bone Graft combines recombinant human platelet-derived growth factor B homodimer (rhPDGF-BB) with a bioresorbable synthetic bonematrix (beta-tricalciumphosphate or β-TCP).

My paper has been widely cited, with a total of 81 citations. For specific citation details, please refer to my google scholar profile. A few notable citations include:

I. Caplan and D. Correa. 2011. “PDGF in bone formation and regeneration: New insights into a novel mechanism involving MSCs”. Journal of Orthopaedic Research. 2011 December; 29(12):1795-1803. – This is an NIH-funded paper that explains how PDGF functions as a powerful therapeutic agent for bone formation and repair. These researchers from Case Western Reserve University (OH, USA) cited my paper and specifically mentioned our study in the context of demonstrating that exogenously delivered PDGF‐BB improved both the rate of bone formation and the bone volume spanning the fracture in rodent models in the presence of diabetes.
Jäger et al. 2011. “Bridging the gap: Bone marrow aspiration concentrate reduces autologous bone grafting in osseous defects”. Journal of Orthopaedic Research. 2011 February; 29(2):173-180. – These researchers from Heinrich-Heine University (Duesseldorf, Germany) ran a study to investigate the potency of bone marrow aspiration concentrate (BMAC) to augment bone grafting and support bone healing. They cited my paper as their 1^st reference. They cited my paper to demonstrated that different orthobiologics such as platelet derived growth factor (PDGF) can promote healing in critical sized bone defects and even in the healing of avascular osteonecrosis.
K Hee et al. 2011. “Augmentation of a Rotator Cuff Suture Repair Using rhPDGF-BB and a Type I Bovine Collagen Matrix in an Ovine Model”. The American Journal of Sports Medicine. 2011; 39(8): 1630-1639. – These researchers from Colorado State University (USA) investigated implanting an interpositional graft consisting of rhPDGF-BB and a type I collagen matrix in an ovine model of rotator cuff repair. They cited my paper to demonstrated that exogenous addition of rhPDGF-BB to animal models of bone injury has shown beneficial effects on tissue repair in vivo, suggesting that augmentation of rotator cuff repair with rhPDGF-BB may lead to improved tendon repair and tendon-to-bone integration

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