Dr. Golub’s laboratory is primarily involved in two research areas: calcification of hard tissues and computer applications in biochemistry and molecular biology. He has been involved in the development of computer programs for prediction and graphical display of protein secondary structure from sequence. In the calcification area, the focus is on the role of the Matrix Vesicles and the enzyme, alakaline phosphotase, in initiating mineral formation in cartilage and bone. His teaching activities include the biochemistry courses for first-year dental students, and lectures to post-doctoral dental students.
Students participating in the School’s student research program can work on the following projects:
The role of matrix vesicles (MV) in hard tissue mineralization has been debated since these enigmatic particles were first observed more than 40 years ago in the matrix of calcifying cartilage. In those studies, and many more recent investigations, it has been shown that MV are present in the matrix of many mineralizing tissues, including cartilage, bone and dentin, and that they are often seen as the site where mineral crystals first form. It is currently believed that MV have two principal roles in initiating calcification: 1) MV enzymes regulate the ratio of Pi to PPi in the extracellular fluid, and 2) MV proteins and lipids, including acidic phospholipids serve as nucleation sites for apatite deposition. Inorganic pyrophosphate (PPi), derived both from hydrolysis of extracellular NTP and from intracellular PPi, inhibits matrix mineralization, but not intravesicular mineral formation. This inhibition is released through the action of Tissue Nonspecific Alkaline Phosphatase (TNAP), which hydrolyzes PPi, thus simultaneously removing the inhibitor and providing additional Pi for mineral formation . Thus, mineralization is said to proceed in two phases: an initial formation of apatite within MV, and a subsequent propagation phase in the matrix. In this formulation, acidic phospholipids and other MV components are thought to nucleate these intravesicular nanocrystals. Subsequently, the intravesicular mineral seeds calcification of matrix macromolecules, principally collagens. When these tissues mineralize, however, the vast majority of the mineral is found associated with collagen fibrils, which are completely permeated and encased in mineral. A key question in the proposed mechanism of MV initiated matrix mineralization is to explain how mineral crystals formed within MV initiate collagen calcification. We hypothesize that MV bind to specific sites on collagen fibrils to facilitate the entry of nanocrystals from MV into the fibrilar structure.
To test this hypothesis, we have been carrying out MV driven collagen mineralization experiments in vitro. MV are isolated from the growth plates of chick tibia by enzymatic digestion and differential centrifugation. Collagen fibrils are formed in vitro by self-assembly. When MV are incubated with the fibrils in a solution containing Ca+2, PO4-2 and beta-glycerol phosphate (BGP), TNAP on the exterior of the MV hydrolyzes the BGP releasing PO4-2 in a manner analogous to that which occurs with PPi in vivo, mineral forms on the collagen fibrils. Analysis by Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDX), Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and Fourier Transform Infrared Imaging (FTIR) reveals that the mineral initially forms in register with the 67 nm ridges which are characteristic of the quarter-staggered collagen fibrillar array, and that this mineral is hydroxyapatite. In contrast, when MV are separated from the collagen fibrils by a dialysis membrane, which allows the Ca+2 and PO4-2 ions to reach the fibrils, but prevents MV from directly contacting the fibrils, the rate, extent and quality of the mineral formed is diminished. Thus, MV mediated collagen mineralization requires physical contact between the MV and the collagen fibrils. The next step in this project is to characterize the molecular binding sites on the collagen fibrils and on the MV.