Graduate School Begins

As a respite from all the research papers, textbooks, and Wikipedia pages that I've been reading, I'd like to talk a little bit about graduate school as it starts to kick into gear. Classes haven't started yet (officially started Thursday, but I have classes on Monday and Wednesday), so I'm not yet encumbered with assignments and deadlines.

I'm currently enrolled in 3 classes and 2 seminars. The classes are:

BIM 173 - Cell and Tissue Engineering

BIM 204 - Physiology for Biomedical Engineers

BIM 284 - Mathematical Methods for Biomedical Engineers

The two seminars are "Meet the Faculty" and a "Distinguished Speaker Series". The faculty seminar involves a different professor each week who talks about his/her research. This is also helpful for current graduate students who are still unsure about whose lab they want to join. Along the same lines, the Distinguished Speaker Series invites guest speakers to present on their current research. The seminars should be very interesting, as it's generally a good idea to gain knowledge in any sort of area, even if it's not immediately pertinent to your specific research goals. It's similar to picking up a book to read. I don't read solely biomedical engineering books. That would be ridiculous. Here's a list of the books I'm currently reading on my Kindle:

1. Storm of Swords - George R.R. Martin
2. East of Eden - John Steinbeck
3. Contact - Carl Sagan
4. Incognito: The Secret Lives of the Brain - David Eagleman
5. The Big Short: Inside the Doomsday Machine - Michael Lewis

That'll actually take me a little while to get through since I won't have that much time for leisure reading (I usually bring out the Kindle now when I'm eating, when I have some downtime, or right before bed). I have 3 fiction books, but even from those you can still learn a lot. Maybe it's not about science (though Contact is heavily science based), but that doesn't mean you can't pick up new things and improve yourself. It could come in handy in future conversation, it could influence your vocabulary and writing style, or serve as a point of inspiration on a project. But mostly you're in it for a great story. Regardless, it's never a detriment to pick up a book to read (unless you really should be doing something else). I read a lot of non-fiction as well. I like reading about new ideas/concepts, memoirs, personal accounts, history, etc. I've had my eye on the John Adams biography by David McCullough for a while now. I might buy it.

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The books I really like (or if they're cheap), I'll purchase. Some are just nice to handle and to have on the bookshelf. Reading on the Kindle, sometimes I forget what it's like to read an actual paperback, i.e. how to flip pages, what real ink looks like on a page. Each format has its merits. I just really appreciate the Kindle for sheer volume and efficiency. I've read many books that I would never have read otherwise.

Some of my literature purchases go towards comic books (it counts as literature). I always feel like I have to defend my interest in comics because people always assume it's nerdy or immature. As I've read in a book recently (Justice: What's the Right Thing To Do? by Michael Sandel), there are different forms of pleasure, but who's to say which is higher than the other? By higher, I mean the more sophisticated, respected, preferred form. The example given in the book was The Simpsons vs. Shakespeare. In a survey, many students say they prefer watching The Simpsons, but rate a Shakespearean work as a qualitatively higher experience. Why is that? Is Shakespeare really that much more worthy and noble than a product of the present? That can be debated. The point is, just because comics may seem like a lesser work, there is a lot underneath the surface that can be just as valuable. I do enjoy the art in comics, I think they are hugely impressive. But I also appreciate the story, the development from issue to issue, the satire, the irony, the social and political commentary. That's what's wonderful about comics, their dual nature and universal appeal. I can read them as an adult, and enjoy them just as much as when I was a kid. As my friend Glenn said, "They're the modern day mythology." It's true. I believe that in order to appreciate literature, you should be able to enjoy them in all its forms. Sometimes, comics are the best medium to get your story or message across, much better than a regular old book would. 

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As I was saying, it's important to read non-fiction as well, to incorporate useful information into your daily life. Maybe it won't impact your research, but maybe it can impact the way you work, the way you live, or how you're motivated. Maybe it'll come in handy when you least expect it. Or, if you're like me, you just read because you have an avid curiosity about everything.

Now that I'm at UC Davis, I think I appreciate the University of Michigan a little more. Michigan's facilities and resources are incredible. And I'm talking about the entire campus. Not to say that I'm severely disadvantaged here, but there are a few things I miss. Most notably, accessible computer labs, fast printing, a centralized campus, lounge/work areas, and an efficient (and free) bus system. It might be that I'm just new here and I haven't discovered everything yet, but I'm having some severe Wolverine nostalgia. I am also not happy with the fact that I have to bike everywhere and cycle through (pun intended) 2-3 shirts a day due to sweating.

This quarter I'll be doing a rotation in a research lab. Hopefully everything works out and I'll be able to stay on but at this point I think it's too early for either of us (me and PI) to commit. So besides classes, I'll be stopping by the lab every now and then to ask questions, read papers, and watch experiments. I'm also hoping to apply for a few fellowships and training grants. This upcoming week will be my first full week in action. There are also several seminars and social events going on so it should be a busy week.

Edit: Also, for those wondering, I've archived all my old posts dating back to a year ago. I still have them, they're just not available for public viewing anymore since I don't think they're relevant within the scope of this blog. I kept the travel posts (to Argentina) because they're a little more interesting.

Bone Remodeling

Building off the last post, I'd like to segue into the mechanism of bone remodeling, or the process of removing old bone (resorption) and laying down new bone. This is a good transition because I already covered the 3 main cell types in bone: osteoblasts, osteocytes, and osteoclasts. Each has an important role in bone remodeling that I briefly touched on previously.

Why do bones need to remodel? 
What we think of when we hear 'remodeling' is a new kitchen, a living room, a basement, an office. A room wears down after continual use, gets old and outdated, and eventually needs a new look. The same sort of idea can be applied to bone matrix. Over time, bones sustain micro-damage and new bone matrix must be produced to maintain skeletal/structural integrity. In the case of injuries, such as fractures, remodeling can also replace bone that is damaged on a larger scale. Physiologically, this process also regulates blood calcium levels. There are 3 phases to bone remodeling: resorption, reversal, and formation.

Resorption: 
The cells responsible for resorption of bone are osteoclasts, which reside on the outer layer of bone. First, the osteoclasts bind to the osteons and induces an infolding of their cell membranes. They secrete enzymes that break down the matrix. This causes calcium, magnesium, phosphate, and collagen to be released into the extracellular fluid. Calcium is transferred into the blood to alter plasma calcium levels.

Reversal:
In the reversal phase, mononucleated cells appear on the surface of the bone or where the "resorption pit" is formed from osteoclast activity. These are most likely mesenchymal stem cells, which proliferate on site and then differentiate into osteoblasts.

Formation:
In the final phase, the osteoblasts fulfill their role by releasing osteoid, eventually hardens into bone matrix. The matrix mineralizes through the help of calcium and phosphorous.

Regulation
Signaling pathways of physiological processes can be intensely intricate and complicated, influenced by a variety of factors. The entire process of bone remodeling is still not fully fleshed out in detail, though much work is being done. Many hormones (parathyroid hormone, calcitrol, etc) and growth factors (TGF-βs, BMP, prostaglandins, etc) regulate bone remodeling. For example, how can osteoblasts form enough bone and tell osteoclasts to stop resorbing bone? After the reversal phase, when osteoblasts are differentiated, the cell presents a protein called receptor activator of NF-kB ligand (RANKL), which then binds to its receptor (RANK) found on osteoclasts. The binding of the RANKL upregulates osteoclast activity, meaning it increases it. Many degenerative bone diseases such as rheumatoid arthritis are caused by overproduction of RANKL (too much resorption of bone). However, osteoblasts can also produce a decoy RANKL protein that binds to the osteoclast receptor and inhibits the osteoclast's ability to bind to osteons and remove matrix, thus downregulating it's activity. Hormones can also affect RANKL (and other protein) expression. The osteoblast thus uses these methods to not only control bone matrix integrity, but also plasma calcium levels.


Sources:
Porter JR, Ruckh TT, Popat KC. Bone Tissue Engineering: A Review in Bone Biomimetics and Drug Delivery Strategies. Biotechnol Prog. 2009, Vol. 25, No. 6. 1539-1560. 
Wikipedia

The Anatomy of Bone

In order to understand the mechanism of osteogenesis, a basic understanding of bone anatomy is necessary. Here, I will provide information gathered from a variety of sources, though I'm keeping everything relatively simple. There are massive repositories of knowledge available on the world wide web that can easily be accessed. I will be showing some illustrations, as it helps to visualize things when it comes to grasping anatomy.

I think it's best to start from a microscopic scale and move upwards to macroscopic. In other words, from cells to tissues, building on each other like bricks of a house. First, why is bone important? Technically, bone is an organ, with the primary function of providing support for the body. However, it also has many other functions, such as protection of internal organs, production of red/white blood cells, mineral storage, release of metabolic factors, etc.

There are three main types of bone cells:

1. Osteoblast - These cells make a protein mixture called osteoid, made up primarily of Type I collagen, that mineralizes to form bone matrix. They also secrete different kinds of enzymes and proteins. Eventually, the immature osteoblast cell matures into an adult bone cell, the osteocyte.
2. Osteocyte - Mature osteoblasts that become trapped in their own secreted matrix proteins. Their functions are to form bone, and maintain matrix and calcium homeostasis.They are also involved in bone remodeling through their mechanosensory receptive properties.
3. Osteoclast - Bone cells that are responsible for bone resorption (removal of bone matrix). Unlike osteoblasts and osteocytes, these are multinucleated cells that work together with the osteoblast to control the amount of bone tissue.

Bone matrix makes up about 1/3 of bone mass, while the other 2/3s is bone mineral. It is made up of organic and inorganic material. The inorganic part is carbonated hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) and the organic is Type I collagen along with various growth factors.The matrix is composed of two types:

• Cortical Bone: Also known as hard compact bone, this is found in the outer shell of bones, surrounding the inner marrow cavities. 
• An osteon is the fundamental unit of cortical bone. The structure of an osteon is a cylinder, involving concentric layers called lamellae. The central region is called the Haversian Canal. Inside the canal are the nerve and blood supplies. The osteocytes in osteons are connected to each other via a network of channels called canaliculi.
• Cancellous Bone: Also known as trabecular or spongy bone. It is less dense and softer than cortical bone. It is found at the end of long bones, in flat bones, and in vertebrae. The bone is highly vascularized, meaning there are lots of blood vessels. The red bone marrow conducts hematopoiesis, or creation of blood cells.
• The trabecula is the fundamental unit of cancellous bone. These are rod-like structures of connective tissue.

These features can be seen in the figure below, taken from Wikipedia.

Sources: 

http://www.uchospitals.edu/online-library/content=P00109

http://cal.vet.upenn.edu/projects/saortho/chapter_01/01mast.htm
Wikipedia

Bone Tissue Engineering

This post is a bit of a "review of a review". It contains information that I've gleaned from an introductory paper in bone tissue engineering (TE). This is a research area I hope to pursue, so I wanted to begin at the basics. I am also learning as I go through the paper, so I will include Wikipedia links to terms that you may want further reading on. This is not exactly a summary, as I have included some of my own thoughts and I don't cover everything, but the scientifically relevant material should be attributed to the authors. The article today is:

Porter JR, Ruckh TT, Popat KC. Bone Tissue Engineering: A Review in Bone Biomimetics and Drug Delivery Strategies. Biotechnol Prog. 2009, Vol. 25, No. 6. 1539-1560. 

Problem: What's the motivation behind bone tissue engineering (or any non-specific regenerative therapy)? Generally, the goal is to restore lost or decreased function of tissue in order to regain normative or near-normative lifestyle. There are several contributors to damaged bone tissue. The most widespread disease factors include osteoporosis, osteoarthritis, and osteomyelitis (osteo- meaning "bone"). Additional factors include physical trauma, orthopedic surgeries, and aging. There is significant need for more efficient and successful therapies as the baby boomer generation reaches retirement age and the population of the elderly rise. New bone TE strategies could also greatly reduce the economic strain of repeated treatment.

Current Methods: Many diseases or injuries do not allow for healing by mechanical fixation alone (setting the bone and letting it self-repair). This results in a non-union, which is a permanent failure of healing. This can occur in traumatic bone fractures, tumor resection, or bone loss, leaving critical-size bone defects. The current method of treatment is autogenous grafting, where host bone is removed from another site (pelvis or iliac) and used to replace the defected area. Allografts from other hosts (cadavers/doners) are also possible, though these carry the risk of infection, disease transmission, or bioincompatibility (immune response leading to rejection).

Bone TE Principles: Currently, synthetic bone scaffolds are used to mimic the physiological environment of the body. More specifically, they must:

• Provide mechanical support
• Have a porous architecture for vascularization and bone growth
• Allow for bone cell migration
• Promote osteogenic differentiation (osteoinduction)
• Enhance cellular activity in scaffold/host integration (osseointegration)
• Degrade in a way that facilitates load transfer to newly created bone
• Not produce toxic byproducts
• Not incite inflammatory response
• Be capable of sterilization
• Deliver drugs in controlled manner

One of the more promising approaches involve the use of mesenchymal stem cells (MSCs). They are multipotent progenitor cells, meaning they have the ability to differentiate into several different types of tissues. For MSCs, this includes osteoblasts (bone), chondrocytes (cartilage), and adipocytes (fat). These MSCs can be seeded onto a synthetic scaffold. While on the scaffold they can be stimulated to differentiate into osteoblasts, which then produce bone extracellular matrix (ECM) ex vivo (outside the body). This scaffold can then be implanted surgically into the defect site. The scaffold then integrates with native tissue.

In this method of bone tissue engineering, there are 3 methods of ex vivo cell culturing: growth factor delivery, bioreactor systems, and gene therapy. Some growth factors that increase osteogenic activity include platelet derived growth factors, bone morphogenic proteins, insulin-like growth factors, and transforming growth factor-βs. These are added to culture media to essentially "feed" the cells. One of the biggest problems in the use of MSCs on scaffolds for implantation is the loss of phenotypic behavior of these cells when placed in vivo (inside the body). This includes osteodifferentiation and the ability to form bone, so viability is a major concern. Another problem is the need for two surgeries, one to remove and harvest MSCs from the host, and another for implantation of the scaffold. Bone also has low concentrations of MSCs and low proliferative capacity, making it difficult to achieve high density on the scaffold. 

This paper focuses on a separate approach to bone TE, involving the use of acellular scaffolds implanted at the defect site. They are infused with drugs and bioactive molecules that are released in a controlled manner as the scaffold degrades. 

• Benefits:
• Easy to sterilize
• Have a shelf life (biodegradable)
• Less prone to infection
• Challenges:
• Design of a scaffold that mimics the micro/nano-scale architecture of bone tissue
• Precise, timed, controlled release of bioactive molecules and drugs
• Match erosion of scaffold with synthesis of bone tissue

Acellular Scaffolds and Bioactive Drug Delivery: There are a few different materials that have been proposed for scaffold construction. Ceramics have been shown to be able to degrade and release drugs in a controlled manner, but have poor mechanical properties. Natural polymers are inherently biodegradable and compatible with native tissue, but they do not have tunable degradation rates (cannot adjust), have poor mechanical properties, and are difficult to sterilize. Synthetic polymers are most successful in controlled release of drugs. These molecules are either covalently bound to the polymers or embedded inside the network matrix. As the polymers degrade, the bioactive molecules are released.

Mechanism of polymer degradation: The polymers have unstable hydrolytic linkage backbones. First, random chain scission occurs, where any ester bond in the polymer has equal chance of being cleaved by hydrolysis (chemical process in which water is broken down into protons and hydroxide ions after breaking apart a polymer). When the molecular weight is reduced to a certain amount (5kDa), the polymers diffuse out of the bulk matrix. Degradation also depends on scaffold architecture, polymerization of multiple polymer types, and the presence of hydrolytic enhancers/suppressors. 

In taking into account scaffold construction and polymer selection, one must focus on: degradation rate compared to bone growth rate, non-toxic degradation products, and ease of use of polymers. I will very briefly cover some commonly used scaffold materials.

Polyesters: The most commonly used polyesters are poly(lactic-acid) (PLA), poly(glycolytic-acid) (PGA), and poly(caprolactone) (PCL). They are FDA approved and have a wide variety of use in medicine, such as in sutures, screws, stents, etc. PLA and PGA have been shown to increase osseointegration and are a good method of slow delivery of drugs. PLA has a low modulus so it must be co-polymerized with a polymer of higher modulus so it can be mechanically stable. PGA alone has a high modulus and degrades completely in 4-6 months. PLA and PCL have longer degradation rates (2+ years). PCL has a high modulus and neutral, absorbable degradation products, making it a good choice for bone TE.

Copolymers: Many researchers combine polymers to create ideal material properties through a method called copolymerization. One of the most common copolymers used is poly(lactic acid-co-glycolic acid) (PLGA). PLGA is good for delivery and encapsulation of drugs, but has poor mechanical properties, so it must be further combined with other materials. 

Growth factor and antibiotic delivery: Bone growth and healing is influenced by a variety of growth factors. However, many scaffolds have only been able to deliver single growth factors, which limit their clinical effectiveness. Research has been shown that controlled and sequential release of multiple bioactive molecules can significantly increase bone regeneration. The challenge is to achieve a highly controlled spatial and temporal delivery method. Often secondary in thought, though equally important, are the pathological factors involved in scaffold implantation. Drugs such as antibiotics, chitin, DNA, RNA, chemotherapeutics, etc, are necessary to prevent infection, or cancer recurrence (in cases of osteosarcoma). Infection can not only inhibit the regenerative process, but can also have fatal consequences. One of the glaring challenges involved with drug delivery is antibiotic viability within the scaffold. 

Challenges and Outlook: Bone TE is promising and could have extensive benefits in several arenas. However, there are also many challenges and improvements that must be made before it can be employed in clinical trials. Researcher must:

1. Create a scaffold that has sufficient mechanical properties throughout the degradative process
2. Effectively release of bioactive molecules and drugs in a highly controlled manner (spatial and temporal)
3. Direct local multipotent cells behavior towards bone regeneration

Next: Through a conglomeration of resources, I hope to address some bone anatomy in the next post.