Determining the Therapeutic Effects of VitB on Type II Diabetic Bone

By Christian Ray

 

 

Introduction

It has been hypothesized that Type 2 Diabetes Mellitus (T2DM)may be a direct cause of osteoporosis due to a decrease in bone tissue quality.^1,3 For example, one study found that T2DM was responsible for a 2-3 times increase of hip fracture risk.¹ While the presence of T2DM was initially thought to be a favorable effect on bone strength since it could increase the mineral density of the tissue, this idea was later reassessed through more detailed and rigorous analysis procedures; furthermore, it has been found that while the amount of bone mass and magnitude of mineral density are relatable to bone strength, the density of diabetic bone does not promote bone toughness and in fact decreases it.^3,8 These procedures, such as micro indentation and computer-generated tomography, revealed that the increase in mineral density had numerous detrimental effects on bone strength including bone remodeling inhibition, increased bone resorption, and overall poorer tissue quality.³

Portrait of Christian Ray

 

To be more specific, bone tissue quality can be deteriorated by non-enzymatic glycation (NEG), a naturally-occurring process resulting in the increase of advanced glycation end-products (AGEs).⁷ The AGEs intra- and inter-fibrillar crosslinks that form throughout the collagen fiber network, causing the matrix to have stiffer properties and thus become more susceptible to breakage; this is a significant factor since over 90% of bone tissue is comprised of collagen fibers which are directly responsible for the elasticity, strength and overall toughness of bone.^7,8,9 From a chemistry viewpoint, the NEG process is the reaction of a reducing sugar (such as ribose) and an amino group (such as lysine and hydroxylysine) from the collagen. In addition, AGE accumulation has also been known to inhibit bone resorption thus leading to a decrease in bone turnover, an important biological process for maintaining bone tissue quality.⁶ Therefore, it is possible that AGEs affect the bone’s mechanical strength by deteriorating tissue quality through the bone resorption process.⁶ Overall, the accumulation of AGEs interferes with the bone structure by degrading the tissue quality and changing its mechanical and chemical properties; furthermore, this process can be further progressed due to aging and diseases such as T2DM.^7-9

 

For this project, together with my peers and mentor Professor Lamya Karim at the Bone Biomechanics Lab at UMass Dartmouth, we aimed to test a drug that can reverse or reduce the harmful effects of AGEs in bone. We decided to utilize the properties of Vitamin B6 (also known as pyridoxamine hydrochloride) due to its ability to counteract AGE formation in bone tissue.^11,12 It is also an ideal treatment due to its natural presence in foods and food supplements, meaning that it is less likely to have harmful side effects unlike previously tested AGE inhibitors. Because of the lack of in-depth mechanical research of vitamin B6 as a treatment for bone deterioration, it is necessary to perform controlled studies on bone treated with varying doses of vitamin B6 in vitro. This will provide useful information to improve treatment for T2D patients.

 

Overall, there is still insufficient research and information regarding the processes and effects of AGEs on bone. The goal of this study is to determine the extent of VitB6 effectiveness as an AGE inhibitor as well as to find any unexpected effects due to the compound.

 

 

Methods

Specimen Collection

All bone samples were collected from the left tibia of 4 female human donors; 3 of these donors had no records of leg-related injuries or bone-affecting diseases and pharmaceuticals, while the fourth donor had Type 2 diabetes. The bone specimens were always shipped frozen with ice packs and insulating boxes and then stored at –20^oC for preservation purposes. Once received, the tibias were each cut into 6 sections using a Craftsman 10” Band Saw where 15% of the total bone was removed from each end and the remaining middle part was divided into four sections. The proximal and distal ends were wrapped in saline-soaked gauze and stored away at -20^oC for other projects. The medial halves of the lateral sections were then cut off using an IsoMet 1000 Precision Saw with a medium-coarse grit diamond blade; the medial halves were then stored in saline-filled zip lock bags and stored at –20^oC for other projects. Using the same saw, each of the lateral halves were then vertically divided into 8-10 cortical beams. Once completed, a South Bay Technology Model 900 Polisher was used to remove the trabecular bone and soft tissue from the beams and to narrow the dimensions down to 2mm x 2mm x 30mm. To maintain awareness of their orientation, the periosteal surface was marked with black ink, and the proximal and distal ends were marked with red and green ink, respectively. The finished beams were then stored in saline-filled microtubes and frozen at –20^oC in cryoboxes.

 

Microcomputed Tomography Imaging

After the beams were done being cut and polished to the correct dimensions, they were then shipped out to Beth Israel Deaconess Medical Center to be imaged via high-resolution Micro-CT to obtain measures of tissue mineral density, cortical porosity, and overall beam geometry.¹⁰ In order to preserve the tissue quality of the samples, the samples were stored in saline in microtubes and cryoboxes and then stored in Styrofoam-insulated boxes with dry-ice.

 

In Vitro Biochemical Incubations

In order to incubate 60 beams in total, the required chemicals for the various solutions were calculated beforehand, as shown in Table 1. Overall, the 60 beams were randomly divided into 5 incubation groups labeled as Control, Ribose, 50mM, 75mM, and 100mM. The Control solution consisted of all the main chemicals aside from the ribose and vitamin B, while the Ribose contained all of the main chemicals aside from vitamin B. The rest of the solutions (which also consisted of the same concentration of ribose) were labeled based on their concentration of vitamin B. The individual amounts of the chemicals in reference to each solution are shown in Table 2.

In order to be more time efficient and ensure consistency of the chemical compositions between the solutions, we mixed all of the ingredients except the ribose and Vitamin B in a 5-gallon glass container with a long, polymer-based, stirring rod. After pouring out enough of this solution into the Control beaker, we added the total amount of ribose into the container and mixed until completely dissolved; once complete, we poured it into the four remaining types of beakers. Then, various amounts of vitamin B were added and dissolved into its respective beakers.

Once the chemicals were all added into the various beakers, the pH of them were individually adjusted using 0.5N NaOH or 0.5N HCl in order to bring the pH within the range of 7.2-7.6; later on, we chose to start using tablets of NaOH due to the rapidly increasing volume from using the diluted version. After the pH was adjusted, the samples were placed in labeled cassettes and randomly put into the five solutions. The beakers were then covered with parafilm and incubated at 37^oC for 14 days, with their pH levels measured and adjusted daily. On the 14^th day, the samples were removed from the beakers, rinsed off with saline, and stored in their respective microtubes with saline and frozen at -20^oC.

Discussion

While most of the project went smoothly throughout the summer, there were some unexpected factors that hindered the projects time efficiency and perhaps other components as well. The first occurrence was when the samples were shipped out to the medical center for Micro CT imaging, where D#00 was accidentally packed and sent to them despite the samples not being complete and ready. This may have caused the samples to be exposed to warmer temperatures than desired for a longer period of time compared to the rest of the donors; however, the samples appeared to be sufficiently cold once shipped back to the lab, indicating that there should be no significant issues relating to tissue degradation.

The second occurrence was when we were adjusting the pH of the solutions during the incubation stage. Initially, we were adjusting the pH using a diluted solution of NaOH; however, the volume increased dramatically due to the drastic changes in pH during the first few days. This could potentially influence the incubation of the samples since the addition of water may have diluted the rest of the chemicals in the solution and thus possibly changing the outcome of its effectiveness.

Overall, the project is still ongoing as there are more stages required for testing and collecting data from the samples that will be conducted throughout the fall semester; this includes mechanical 3-point bending tests, micro indentation, cRPI, and fAGE measurements.

 

References

1. Karim L, Bouxsein ML. Effect of type 2 diabetes-related non-enzymatic glycation on bone biomechanical properties. Bone. 2016;82:21-27. doi:10.1016/j.bone.2015.07.028
2.  Valcourt U, Merle B, Gineyts E, Viguet-Carrin S, Delmas PD, Garnero P. Non-enzymatic glycation of bone collagen modifies osteoclastic activity and differentiation. J Biol Chem. 2007;282(8):5691-5703. doi:10.1074/jbc.M610536200
3. Wongdee K, Charoenphandhu N. Update on type 2 diabetes-related osteoporosis. World J Diabetes. 2015;6(5):673-678. doi:10.4239/wjd.v6.i5.673
4. Singh, R., Barden, A., Mori, T. et al. Advanced glycation end-products: a review. Diabetologia 44, 129–146 (2001). https://doi.org/10.1007/s001250051591
5. Karim L, Moulton J, Van Vliet M, Velie K, Robbins A, Malekipour F, Abdeen A, Ayres D, Bouxsein ML. Bone microarchitecture, biomechanical properties, and advanced glycation end-products in the proximal femur of adults with type 2 diabetes. Bone. 2018;114:32-9. Epub 2018/06/02. doi: 10.1016/j.bone.2018.05.030. PubMed PMID: 29857063; PMCID: PMC6141002.
6. Karim L, Vashishth D. Heterogeneous glycation of cancellous bone and its association with bone quality and fragility. PloS one. 2012;7(4):e35047. doi: 10.1371/journal.pone.0035047. PubMed PMID: 22514706; PMCID: PMC3325937.
7. Poundarik AA, Wu PC, Evis Z, Sroga GE, Ural A, Rubin M, Vashishth D. A direct role of collagen glycation in bone fracture. Journal of the mechanical behavior of biomedical materials. 2015;52:120-30. Epub 2015/11/05. doi: 10.1016/j.jmbbm.2015.08.012. PubMed PMID: 26530231; PMCID: PMC4651854.
8. Tang SY, Zeenath U, Vashishth D. Effects of non-enzymatic glycation on cancellous bone fragility. Bone. 2007;40(4):1144-51. doi: 10.1016/j.bone.2006.12.056. PubMed PMID: 17257914; PMCID: PMC4398019.
9. Vashishth D, Gibson GJ, Khoury JI, Schaffler MB, Kimura J, Fyhrie DP. Influence of nonenzymatic glycation on biomechanical properties of cortical bone. Bone. 2001;28(2):195-201. PubMed PMID: 11182378.
10. Research centers. BIDMC of Boston. https://www.bidmc.org/research/research-centers. Accessed September 8, 2022.
11. Voziyan PA, Khalifah RG, Thibaudeau C, Yildiz A, Jacob J, Serianni AS, Hudson BG. Modification of proteins in vitro by physiological levels of glucose: pyridoxamine inhibits conversion of Amadori intermediate to advanced glycation end-products through binding of redox metal ions. J Biol Chem. 2003 Nov 21;278(47):46616-24. doi: 10.1074/jbc.M307155200. Epub 2003 Sep 15. PMID: 12975371.
12. Abar O, Dharmar S, Tang S. The effect of aminoguanidine (AG) and pyridoxamine (PM) on ageing human cortical bone. Bone & joint research. 2018;7(1):105-10.