Internship at FACTS: A Histological Study of Monkeys
I conducted an internship at the Forensic Anthropology Center at Texas State (FACTS) in the fall of 2017 at both the Osteological Research and Processing Laboratory (ORPL) and the Grady Early Forensic Anthropology Research Laboratory (GEFARL) facilities. The goal of the work conducted at these facilities is to further the research on biological aspects of humanity through examination of skeletal remains. I had the opportunity to work on a histology project with the FACTS coordinator Sophia Mavroudas to study osteons and Haversian canals in monkey bones. Sophia is interested in comparing the drifting osteons and Haversian canal sizes in monkey histological samples with human histological samples. Currently, there are no methods to differentiate human bone from monkey bone and Sophia is conducting research to produce a method for doing so. The portion of the project I worked on included learning techniques for processing bones, taking inventory, taking photographs, using Photoshop, using a micro-CT scanner and learning how to take histological samples. These techniques and skills are applicable to both forensic anthropological and bioarchaeological contexts.
Histology is the study of the microstructure of tissues, in this case bone, and how the tissue communicates and adapts to physical changes (De Boer and Van der Merwe 2016). Bone tissue is constantly changing, and metabolic changes can cause increased osteoclastic and osteoblastic activity, or bone destroyers and bone builders, respectively. Osteons are the structural unit of compact, or mature, bone that form during the process of bone remodeling. Primary osteons are formed by the mineralization of cartilage and secondary osteons are formed by the replacement of existing bone. The formation of secondary osteons begins when osteoclasts destroy woven, or new, bone, hollowing out a narrow passage called a Haversian canal. A Haversian canal is a small hollow channel in the center of an osteon that contains blood vessels that carry blood to the bone. After the osteoclasts create the channel, osteoblasts lay down new bone on the sides of the Haversian canal, entrapping the osteoclasts within the small spaces of the lamellae. The lamellae are the concentric layers of compact bone that form around the Haversian canal. The final layer of compact bone forms a boundary, called a cement line, that can be observed as a scalloped edge under a microscope. After being trapped in the lamellae, osteoclasts turn into osteocytes, or mature bone cells, that direct the osteoclasts and osteoblasts to remodel bone to maintain the bone structure. When the process of forming new Haversian canals starts, the osteons overlap and are called fragmentary osteons. A drifting osteon is an osteon that drifts in one direct or changes directions multiple times, leaving behind a trail of lamellae.
Choosing the correct technique when processing remains is paramount in avoiding the risk of damaging the bones prior to examination. When I received the baboon (Papio hamadryus, see Fig. 1), which had been donated to FACTS by the University of Texas, it had already been dissected. Typically, the donated bodies used at the Forensic Anthropology Research Facility (FARF) at Texas State University are fully fleshed when they are received. The bodies are left outside to decompose for research purposes and are then taken to ORPL, where they are macerated and cleaned by volunteers. Since a large portion of the flesh on the baboon had been removed and the joints were previously disarticulated, I skipped the decomposition process and attempted to macerate the monkey with the same technique used on the donated bodies. The decomposition process would have taken too long to ensure completion of this project in one semester and, because I was not conducting any research on the process of decomposition, there was no need to leave the baboon outside to putrefy. With help of the ORPL supervisor, Kari Helgeson, I macerated the baboon in a crockpot filled with boiling water and Tergazyme to soften the tissues.
The objective of this process is to break down the tissues so that it will be easier to utilize metal tools such as a dental scraper and remove the remaining flesh from the bones. After a few attempts to macerate the baboon, we realized that the tissue had been treated with some sort of preservative to prevent decomposition. We eventually reached a point where it was too risky to continue macerating the baboon and I had to remove as much flesh as possible with the metal tools. Over-boiling bones can cause the bones to absorb the water and become soft, which may permanently damage them and even cover up any trauma on the outer surface of the bone (Mann and Berryman 2012). Sophia attempted to get access to dermastid beetles that can eat away at the flesh without damaging the bone, even if the flesh is treated with a preservative. However, FACTS has been trying to get these beetles for a while without any luck and, unfortunately, we did not obtain any beetles. Attempting to remove the remaining flesh with metal tools was my only option at this point, but even this method has its drawbacks. The metal scraping tools can damage the surface of the bone, and the damage may be confused with perimortem (around time of death) or postmortem (after time of death) trauma which would need to be explained in a forensic setting (Mann and Berryman 2012). Despite this obstacle, for the purposes of this project, minor postmortem scrapes on the surface of the bone will not interfere with the research being conducted.
Inventorying the bones provides the opportunity to examine trauma, pathology and demography of the individual. Human and baboon skeletons have nearly identical skeletal elements, and therefore the Standards for Data Collection from Human Skeletal Remains by Buikstra and Ubelaker (1994) was the protocol followed for inventorying the baboon. Keeping a detailed record of the remains provides a way to keep track of which skeletal elements are absent or present and which elements are fragmentary.
When making an inventory list, the observer should be extremely detailed in describing the trauma or pathological lesions found on the bones. If the observer is not detailed when describing the bones, it is possible that things could either be overlooked or trauma to the bones made after the inventory is done could be mistaken for perimortem or postmortem trauma. In the forensic context, this mistake could possibly throw off an investigation. In bioarchaeology, inventory lists are important when determining the minimum number of individuals found at a burial site as well as establishing a biological profile. A biological profile is the age, sex, stature and race of an individual, and, although one can attempt to establish this during skeletal inventory, it is impossible to establish a concrete profile until skeletal measurements called morphometrics are taken. For the purposes of this project, we did not need to establish a biological profile by conducting a morphometric analysis.
Photography and Photoshop
The use of photography and photo editing in biological anthropology is important for providing an analysis on current projects. Photographing the baboon skeleton was important to this project for record keeping purposes. Some of the bones were going to be used for histological samples, which requires destroying the bone. But in some cases, photographs can be used for documentation for safety precautions in case the bones are accidentally destroyed. Additionally, these photographs may be sent to other anthropologists to help analyze the lesions found on the skeleton or to attempt to help establish a biological profile, in which case the bone measurements may be sent with the photographs. The purpose for Photoshop in this project was to remove the shiny background caused by the semi-reflective tables upon which the skeletons were photographed. Normally, a black cloth would be put underneath the skeleton to contrast the color of the bone so that it stands out and is easier to observe. However, GEFARL is in the process of moving to a new facility and the black cloth was nowhere to be found. Photoshop is acceptable in bioarchaeology, but when it comes to forensic cases, photo editing pictures will raise suspicions and render the photographic evidence inadmissible in court. Documenting the skeletal elements through photography provides an objective way for many people to continuously examine skeletal remains if they were to be destroyed, whether it be by accident or on purpose. Photographs can be hard to interpret, which is why Micro-CT-scans of bones can be taken to further preserve the structure of the bone.
Micro-CT Scanner X5000
Scans taken of dry skeletal remains by a Micro-CT Scanner can provide a detailed copy of bones that can be 3D printed or viewed virtually for in depth examinations of skeletal material that are either not present or that have been destroyed for procedures such as histology samples. A Micro-CT scanner, or Micro-Computed Tomography scanner is an x-ray imaging technique that takes 360 degrees of x-rays of an object from every angle. The x-ray generator sends x-rays through an object which is picked up by a recorder on the other side to make a slice-by-slice image of the bone (www.bruker-microct.com/company/methods.htm). The scan allows you to see the microstructure of an object without destroying the object by either looking at and interpreting the scan on the computer screen, or by 3D printing the object and cutting into the printed copy instead of the object.
The Micro-CT scanner used to scan the femora of the monkeys was the Micro-CT Scanner X5000 at GEFARL (Fig. 2). Dr. Cunningham and only one other person at Texas State University are authorized to use this complicated machine and you must undergo a myriad of technical courses to operate it. The machine is huge, but the area where the scan is taken can only hold one large bone or two to three small bones at most. This technology is important in cases where the bone, or other objects such as artifacts from archaeological sites, cannot be destroyed. Because the histology project I was working on required destroying the monkey femora, CT-scans needed to be taken to preserve a 3D image of the bone. Unfortunately, because of how expensive and time-consuming 3D printing is, we did not 3D print the monkey femora. The downside to using a Micro-CT scanner and 3D printer is how costly it is; most universities do not own either of these technologies and cannot carry out experiments that require CT-scans.
Figure 2: Right baboon femur next to Mirco-CT Scanner X5000.
The practice of histology is becoming more important as studies reveal its vital role in understanding how the environment, metabolism and genetics influence bone micro-structure. Sophia Mavroudas has a protocol used by many of her anthropology colleagues who do histology. First, I cut a section of the monkey femur at the midshaft with a Dremel saw and embedded the section of bone in resin to make a histoblock (Fig. 3). Once this histoblock was dry, I cut thin sections of the histoblock called wafers using the Isomet 1000 Precision saw (Fig. 4). I then ground down the wafers with the Metaserv 3000 Variable Speed Grinder-Polisher (Fig. 5) so that they were thin enough to see light through the bone. Once the wafers were thin enough, I mounted them onto slides so that a cross-section picture (Fig. 6) could be taken through an Olympus DP27 microscope with a program called cellSens (Fig. 7). From there, any type of study dealing with osteons can be done.
In this project, I counted the primary osteons, fragmentary osteons and drifting osteons and measured the area, perimeter and circumference of the primary osteons and Haversian canals through the ImageJ program. Histology samples can tell a lot about disease progression, postmortem microstructural changes due taphonomy and the demographic of an individual. By observing the changes made by osteoclasts and osteoblasts, age, sex, race and general health of the individual can be determined. The field of histology is new and continuously growing and new discoveries on what bone microstructure can tell us will be made.
Histology can help to compare the microstructure of monkey and human bone to help differentiate between the two in both archaeological and forensic contexts. There have not been many studies conducted that compare the size and frequency of osteons in human bone, and the variables that affect the size differences are not well understood. By comparing histological samples of monkey bone to samples of human bone, one can understand how age, sex, race and environment of an individual can act on and drive variation in remodeling. This study will shed light on how bone grows and develops, giving us insight into how the individual lived.
The Forensic Anthropology Center at Texas State (FACTS) is an amazing facility that provides an array of opportunities and experiences for students to explore the many sub-disciplines of biological anthropology. Additionally, volunteering at the new Grady Early Forensic Anthropology Research Laboratory (GEFARL) provides students with an opportunity to work with the hundreds of skeletons in the Texas State University Donated Skeletal Collection to learn more about human variation and skeletal biology. This internship has taught me many new skills that I will be able to apply to any branch of biological anthropology, no matter which route I go. I have also gained a new fondness for histology that I never imagined I would have a few months ago. I will be able to take these new skills I have and apply them to graduate school and possibly even come up with a thesis based on histology. Biological anthropology serves to study the biology of humans through the lens of culture, social sciences, behavior and evolution. Whether I go on to give a voice to populations of the past or to help solve criminal investigations, I aim to use my experience as an anthropologist to help people.
De Boer HH, Van der Merwe, AE. 2016. Diagnostic dry bone histology in human paleopathology. Clinical Anatomy: 29:831-843.
Buikstra JE, Ubelaker DH. 1994. Standards for data collection from human skeletal remains. Arkansas Archaeological Survey Research Series No. 44.
Mann RW, Berryman HE. 2012. A method for defleshing human remains using bleach. J Forensic Sci: 57(2):440-442.