THE WAKE of the GROWTH PLATE a Thesis

Home , Ossification center

BONE GROWTH: THE WAKE OF THE GROWTH PLATE

A thesis submitted

To Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Arts

Samantha H. Magrini

August 2021

Except for previously published materials

Thesis written by

Samantha H. Magrini

B.A., Youngstown State University, 2017

M.A., Kent State University, 2021

Approved by

______, Advisor Linda Spurlock

______, Chair, Department of Anthropology Mary Ann Raghanti

______, Interim Dean, College of Arts & Sciences Mandy Munro-Stasiuk TABLE OF CONTENTS ------iii

LIST OF FIGURES ------iv

LIST OF ABBREVIATIONS ------v

ACKNOWLEDGEMENTS ------vi

CHAPTERS

I. INTRODUCTION ------1

Wolff’s Law: History and Overview ------1

Limb Bud Formation ------4

Endochondral Ossification ------6

Proposed Research Hypothesis ------11

II. METHODS ------12

The Libben Osteological Collection ------12

Sample Selection ------14

Three-Dimensional Scanning and Printing ------15

Final Processing for Analysis ------18

Ill. RESULTS ------21

Image Overlays ------21

CONCLUSIONS ------25

REFERENCES ------27

iii LIST OF FIGURES

Figure 1. Dorsal View of the Limb Bud ------5

Figure 2. Schematic Layout of the Growth Plate ------7

Figure 3. The PTHrP-Ihh Negative Feedback Loop ------9

Figure 4. 3D Scans of Specimen 10063 ------16

Figure 5. 3D Scans of Specimen 03210 ------17

Figure 6. 3D Scans of Specimen 02062 ------17

Figure 7. Comparison of Specimen #02062 and the 3D Printed Replica ------18

Figure 8. Cross-Sectioning of the 3D Printout ------19

Figure 9. Specimen #02062 Alongside Comparison Specimens ------19

Figure 10. Side-by-Side Comparisons of Specimen #12206 and Slice 1 ------21

Figure 11. Overlay Comparisons of Slice 1 and Specimen #12206 ------21

Figure 12. Overlay Comparisons of Slice 2 and Specimen #3203 ------22

Figure 13. Overlay Comparisons of Slice 3 and Specimen #5035 ------22

Figure 14. Overlay Comparisons of Slice 4 and Specimen #4055 ------22

Figure 15. Overlays of Proximal Growth Plate Surfaces ------23

iv LIST OF ABBREVIATIONS

Abbreviation Meaning Page

AER Apical ectodermal ridge 4

AP Anterior-posterior 5

BMP Bone morphogenetic protein 4

FGF Fibroblast growth factor 4

Hox Homeobox 5

Ihh Indian hedgehog 8

LRP Lipoprotein receptor related protein 6 mRNA Messenger RNA (ribonucleic acid) 10

PTHrP Parathyroid hormone-related protein 8

PZ Progress Zone 5

Runx2 Runt-related transcription factor 2 6

Shh Sonic hedgehog 4

Sox9 SRY-Box transcription factor 9 6

ZPA Zone of polarizing activity 4

v ACKNOWLEDGEMENTS

I would like to start by thanking my advisor, Dr. Linda Spurlock. You saw something in me from day one, and I have so enjoyed traversing the highs and lows of this process with you. I couldn’t have made this happen without your help and support. My other thesis committee members Dr. C. Owen

Lovejoy, and Dr. Richard Meindl, thank you for your encouragement and input in making this a reality. It has been an educationally eye-opening experience to have you give your thoughts and advice throughout.

I also would like to acknowledge the help and support of Dr. Mary Ann Raghanti, Chair of the

Anthropology Department, who not only provided needed critique of my early chapters, but who after many emails aided in setting up connections with the Design Innovation Hub for me.

I would like to thank the Kent State Design Innovation Hub, specifically Andrea Oleniczak and

J.R. Campbell for making it possible for me to access the 3D scanning equipment, especially on such short notice. Your help along with that of the student workers introduced me to the amazing possibilities which are presented when departments come together and form interdisciplinary connections. Most notably within the DI Hub student workers I must thank Aishwarya Yelakonda for all the technical support and help with ‘Eva’. I couldn’t have made this happen without her, and I thoroughly enjoyed the time we spent together as she helped me traverse the technical side of things.

Closer to home, yet across many miles, I must thank my parents, Brian, and Teresa Carley. I wouldn’t have been able to make this happen without the constant support and encouragement they have given me every step of the way.

Finally, I need to thank my husband, Troy Magrini Jr. He has been my rock throughout the highs and lows of this process. I have known from day one that he would be there to keep me going on the tough days, and to cheer for me on the good ones. I am so lucky to have him there to help me see what awaits on the finish line.

INTRODUCTION

Wolff’s Law: History and Overview

It is not uncommon for many to link bone growth and development to the effects of external mechanical loading forces, with the belief that loading is the primary driving factor of bone formation

(Bertram and Swartz 1991; Frost 1983; Jansen 1920; Pearson and Lieberman 2004; Ruff, Holt, and

Trinkaus 2006; Villemure and Stokes 2009). These hypotheses can be linked back to German anatomist

Julius Wolff, who in 1882 produced several self-ascribed ‘laws’ regarding the formation of bone from which many have drawn inspiration (Wolff 1892; Wolff 1886). He stated that “the law of bone remodeling is the law according to which alterations of the internal architecture clearly observed and following mathematical rules, as well as secondary alterations of the external form of the bone following the same mathematical rules, occur as a consequence of primary changes in the shape and stressing or in the stressing of the bones,” (Wolff 1892; Wolff 1886).

Since Wolff’s influential treatise, many others added to his hypothesis. These include Young’s

Modulus of Elasticity, an engineering principle which has since been applied to the properties of bone, and which considers bones’ elastic properties (Pearson and Lieberman 2004). Young’s Modulus as applied to bone specifies a range of stress-strain which bone can withstand without breaking and still return to its original size and shape (Pearson and Lieberman 2004). Another is the Hueter-Volkmann Law of mechanical modulation of bone growth, which suggests that immature bone length is dictated by compressive forces acting upon the epiphyseal cartilage (Villemure and Stokes 2009; Volkmann 1865;

Walker et al. 2002). Increases in pressure inhibit bone growth, whereas decreases in pressure and tension stimulate bone growth (Hueter 1862a; 1862b). This interesting seems to contradict many of the principles applied beneath ‘Wolff’s Law’, as it suggests that bone growth will be inhibited by pressure, rather than the typical claim of these ‘Laws’ which suggest increased stress and strain will increase bone mass. Wolff

1 also worked alongside Wilhelm Roux, with whom he developed the “doctrine of the functional form of bones,” which states that the structure and form of bones is determined entirely, “mechanically” and

“mathematically”, by tension and pressure stresses (Jansen 1920; Wolff 1892). Roux believed that the bone cells were capable of responding to mechanical stress and therefore were responsive to loading

(Ruff, Holt, and Trinkaus 2006). Taken together these ideas have contributed to what is now known as

‘Wolff’s Law’ (Pearson and Lieberman 2004).

The main principle of Wolff’s Law is that bone adapts to mechanical loading forces, and that there is a functional morphology related to bone form and function (Pearson and Lieberman 2004).

Essentially, bone would be laid down where needed and removed where not (Walker et al. 2002). Cortical bones would be expected to increase in volume as a response to force, which would increase the bone’s loading capacity (Bertram and Swartz 1991). Wolff and his followers believe that bone’s primary formation is directed by loading pressures, and that bone evolves towards optimization to reduce risk of fracture (Cristofolini et al. 2013; Frost 1983; Jansen 1920).

Wolff et al. declared that these mechanical forces that are driving bone growth, through some unknown process, are adaptations to modify bone to prevent bending and potential fracture (Bertram and

Swartz 1991; Carter et al. 1987; Cristofolini et al. 2013; Frost 1983; Pearson and Lieberman 2004).

Although this may be partially true for trabecular bone, there is no evidence that this is true for cortical bone, nor is there sufficient evidence that this purported adaptation increases an organism’s fitness

(Bertram and Swartz 1991).

In their paper The ‘Law of Bone Transformation’: A Case of Crying Wolff? (1991), Bertram and

Swartz outlined many of the failures of Wolffian researchers to definitively prove that mechanical loading results in the stimulation of bone growth and development. They point out that many hypotheses have been supported via in vivo testing, with the problem being that this does not represent natural responses

(Bertram and Swartz 1991). In vivo testing has been conducted in pigs, rabbits, dogs, and sheep, to name a few examples (Chamay and Tschantz 1972; Goodship, Lanyon, and McFie 1979; Hert, Liskova, and

Landrgot 1969; Lanyon et al. 1982; Moreland 1980). In each instance the experimental design required

2 removal of bone, and/or the surgical implementation of strain gauges on bone. In the case of Lanyon’s study for example, the experimentation required removal of the ulna in mature sheep to measure the increase in walking strain on the radius (Lanyon et al. 1982). During the Chamay and Tschantz testing they resectioned the radial diaphysis of one group of dogs, and in another performed a radial osteotomy with the introduction of pins through the olecranon and distal ulna with the addition of an external compression device to facilitate loading (Chamay and Tschantz 1972). These two examples show that surgical experiments go far beyond the natural conditions under which bone would normally be loaded, putting into question how useful the results are for showing the biological response of bone to force.

Further to that though, surgical experimentation can cause extreme reaction from bone tissue, even at anatomical locations removed from the site of investigation (Bertram and Swartz 1991). Essentially this extreme response could render all in vivo results null, leaving no quantitative answer to the effect of loading force upon bone.

Not only may these in vivo results be misleading due to the nature of the tests themselves, they often fall short of accomplishing their goal of supporting Wolff’s Law. It has been demonstrated that no type of cortical remodeling takes place through rotation, and that expected changes in growth rate from dynamic loading of the growth plate remain unchanged (Moreland 1980; Villemure and Stokes 2009). It can also be argued that bone formation occurs regardless of force, as seen in individuals who suffer some type of childhood paralysis (Jansen 1920). Additionally, for mechanical loading to have an effect, the loading would have to exceed pressures which are ordinarily exerted by the weight of the body, meaning that it likely would only have an effect in extreme circumstances, not as part of the underlying formative process (Jenkins 2008).

It is very concerning that given a lack of solid evidence to support claims tied to Wolff’s Law, it has become an almost unquestioned tenet of biology (Bertram and Swartz 1991). Wolff’s Law is invoked throughout paleontology and bioarcheology, as well as numerous other skeletal developmental disciplines

(Ruff, Holt, and Trinkaus 2006). Another concern is that these Wolffian paradigms seek to prove that morphology is adaptive. Given that adaptation requires some type of influence on an organism’s fitness

3 and ability to survive and reproduce, there is no evidence that any tenet of Wolff’s paradigm would meet these standards (Gould et al. 1979; Pearson and Lieberman 2004). Instead, they appear to be chasing after

‘spandrels’, which are simply byproducts of evolutionary development (Gould et al. 1979).

Evidence from the field of genetics has demonstrated that coordinated growth is an exceptionally complex process, requiring an almost simultaneous amalgam of cell migration, proliferation, differentiation, and apoptosis, controlled through a plethora of hormones and regulator genes (Burdan et al. 2009; Carroll, Grenier, and Weatherbee 2013; Jenkins 2008; Karimian, Chagin, and Sävendahl 2012;

Karsenty 2003; Kronenberg 2003; Kronenberg 2006). So, for many to still focus their attention on finding a purely external mechanical answer to bone development is not only misguided, it detracts from studies which could be progressing our collective knowledge.

Numerous genes and hormones are involved in bone’s growth and development, which include

“bone morphogenetic proteins (BMPs), Wnts, fibroblast growth factors (FGFs), hedgehog proteins, insulin-like growth facts, and retinoids,” as well as, “growth hormone, thyroid hormone, oestrogen, androgen, vitamin D, and glucocorticoids,” (Kronenberg 2003, 332).

Limb Bud Formation

The embryonic limb bud begins with a proliferation of mesenchymal cells (Carroll, Grenier, and

Weatherbee 2013; Karsenty 2003). This proliferation requires signals from fibroblast growth factor 10

(FGF10) to enlarge the group of cells below the epidermis through mitotic division (Carroll, Grenier, and

Weatherbee 2013). As growth continues the mesenchymal cells induce the ectoderm to express FGF8 which will eventually form the apical ectodermal ridge (AER), the thickened ridge of the limb bud where the dorsal and ventral halves meet (Figure 1.) (Capdevila and Belmonte 2001; Carroll, Grenier, and

Weatherbee 2013; Karsenty 2003). The AER subsequently signals FGF8 for continued proliferation and differentiation. The zone of polarizing activity (ZPA) which controls patterning of the anterior-posterior margin of the limb bud is another organizing region, and one which is the source of Sonic hedgehog

(Shh), one of the hedgehog proteins (Carroll, Grenier, and Weatherbee 2013). The AER and ZPA act

4 together creating a circuit in which FGF and Shh expression drive the development of the limb bud

(Carroll, Grenier, and Weatherbee 2013; Karsenty 2003).

AER

Proximal PZ Distal

ZPA

Posterior Figure 1: Dorsal View of the Limb Bud: comprised of mesenchymal cells within the ectoderm. The Zone of Polarizing Activity (ZPA) patterns the growth of the Anterior-Posterior (AP) Axis, and the Apical Ectodermal Ridge (AER) directs outward growth whilst keeping undifferentiated mesenchymal cells of the Progress Zone (PZ) in their undifferentiated state Adapted from: Capdevila and Belmonte 2001, 90

Once the ZPA and AER are formed, the proximal mesenchymal cells begin to form pre-skeletal condensations, which will become the hyaline cartilage models from which endochondral bone formation begins (Carroll, Grenier, and Weatherbee 2013). The mesenchymal condensations form in proximal to distal order, and require Hox genes to determine patterning and formation (Carroll, Grenier, and

Weatherbee 2013). The Hox (homeobox) genes are a family of genes that regulate development (White,

Black, and Folkens 2011). There are three phases to Hox expression, again working from most proximal to most distal (Carroll, Grenier, and Weatherbee 2013). The first phase of Hox expression is associated with development of the most proximal limb segments, the upper arm and leg, with the most anterior position of Hox6 expression determining the position of the forelimb buds (Carroll, Grenier, and

Weatherbee 2013). Phase two of Hox expression occurs in the forearm and lower leg, the next most proximal features (Carroll, Grenier, and Weatherbee 2013). The third phase deals with the most distal elements, the wrist, hand, ankle, and foot (Carroll, Grenier, and Weatherbee 2013).

5 The osteochondroprogenitor cells of the mesoderm will differentiate into both the chondrocytes, cartilage-forming cells, and osteoblasts, bone-forming cells (Hall 2015; Karsenty 2003). Chondrogenesis, requiring genes encoding both growth factors and transcription factors, will result in the differentiation of chondrocytes into resting, proliferating, and hypertrophic forms, all found within the growth plate

(Karsenty 2003). It is also worth pointing out that the chondrocyte, in its hypertrophic form is one of the principle driving factors of bone growth (Kronenberg 2003). Osteoblasts require Runx2 and Osterix for differentiation, and the low-density lipoprotein receptor-related protein 5 (LRP5) signaling pathway for proliferation (Karsenty 2003; Kronenberg 2003). There are also a series of transcription factors which control for osteoclast differentiation, the cells responsible for resorption of bone (Hall 2015; Karsenty

2003). Once the cartilage model is formed, ossification will follow.

Endochondral Ossification

Endochondral ossification is a multistep process that starts after the condensation of mesenchymal cells. SOX9 expression works to convert mesenchymal cells of the condensation into chondrocytes, which will form a collagen matrix; the avascular hyaline cartilage anlage, and the perichondrium, the fibrous connective tissue covering cartilage, which will differentiate into the periosteum, the connective tissue covering bone (Hall 2015; Kronenberg 2003; 2006; Walker et al. 2002).

The mesenchymal cells of the deepest layer of the periosteum will differentiate; osteoblasts will start to synthesize osteoid tissue around the exterior of the cartilage model forming a bony collar (Kronenberg

2006; Walker et al. 2002). The bony collar is the first bone to be laid down around the periphery of the cartilage model (Walker et al. 2002). The cells at the center of the anlage undergo apoptosis and calcification, with the hypertrophic chondrocytes directing mineralization of the matrix (Kronenberg

2006; Walker et al. 2002). Shortly following is the introduction of the nutrient artery into the diaphysis, once again stimulated by the chondrocytes (Kronenberg 2006; Walker et al. 2002).

Vascularization brings osteoprogenitor cells to the center of the cartilage model, where they differentiate into osteoblasts which begin to form bone (Walker et al. 2002). At the same time, chondrocytes in the center of the anlage continue to apoptose, during which those in the center

6 hypertrophy and coalesce into flattened spicules (Walker et al. 2002). Following the formation of spicules, the remaining cartilage calcifies (Walker et al. 2002). The osteoblasts lay down bone over the calcified cartilage left from the hypertrophied chondrocytes, and it is this which creates the primary center of ossification (Walker et al. 2002). That primary center of ossification then spreads as more osseous material is laid down (Walker et al. 2002). Formation of the primary ossification center means that the medullary cavity begins to form (Walker et al. 2002). As more bone is laid down on both the bone collar and around the marrow cavity further calcified matrix is left behind (Walker et al. 2002). Osteoclasts respond by removing older matrix, enlarging the medullary cavity along the shaft of the bone (Walker et al. 2002).

Whilst these processes are taking place the bone grows in length and diameter. The chondrocytes continue to proliferate, forming columns, and eventually hypertrophy (Kronenberg 2006). It is through the increase in these chondrocyte numbers that the bone length extends (Kronenberg 2006). Eventually the secondary ossification centers take shape in the epiphyses at the distal and proximal ends of the bone,

Figure 2: Schematic Layout of the Growth Plate: (Line of recent fusion marked on scan of specimen #02026) showing the three zones of chondrocyte activity: Resting, Proliferative, & Hypertrophic. Adapted from Karimian, Chagin, and Sävendahl 2012, 2

through similar processes of development and vascularization as the primary ossification center (Walker et al. 2002). Once the epiphysis forms, the epiphyseal plate, a plate of hyaline cartilage, remains between the between the diaphysis and epiphysis (Walker et al. 2002). This epiphyseal growth plate will continue the bone’s longitudinal growth through post-natal life and in itself is controlled by a range of genetic factors (Kronenberg 2006).

The epiphyseal growth plate is comprised of four main zones, each of which contains chondrocytes (Figure 2.) (Burdan et al. 2009; Karimian, Chagin, and Sävendahl 2012; Walker et al.

2002).The zones: resting, proliferative, hypertrophic, and the zone of bone formation are regulated by both hormones and growth factors (Burdan et al. 2009; Karimian, Chagin, and Sävendahl 2012;

Villemure and Stokes 2009). The resting zone is characterized by low levels of chondrocyte proliferation, and is found closest to the epiphysis (Burdan et al. 2009; Walker et al. 2002). This first layer merges into the second, the proliferative zone where chondrocytes are actively mitosing, flattening, and dividing into columns (Burdan et al. 2009; Walker et al. 2002). These cells produce the collagen matrix which will be used as the framework onto which osseous material will later be calcified (Burdan et al. 2009; Walker et al. 2002).

Below the proliferative zone is the hypertrophic zone, and as the name suggests here the chondrocytes grow in size, and undergo cell death (Burdan et al. 2009; Walker et al. 2002). This means that there is a reduction in cell division. Also at this level alkaline phosphate is being produced which aids in widening the growth plate (Burdan et al. 2009). At the end of the growth plate closest to the diaphysis calcification occurs to the cartilage (Burdan et al. 2009; Walker et al. 2002). At the very base of the growth plate the forth zone exists, the zone of bone formation (Walker et al. 2002). This is where osteoblasts begin to lay down osseous material over the calcified cartilage, producing the final bone

(Walker et al. 2002). These zones, and the processes which take place within them, are controlled by a multitude of hormones and growth factors.

8 Chondrocytes and periochondrial cells at the ends of the developing bone synthesize parathyroid hormone-related protein (PTHrP) (Kronenberg 2006). PTHrP causes the chondrocytes to continue proliferation, encouraging growth, whilst delaying them from going into hypertrophy (Kronenberg 2003;

Kronenberg 2006). When the chondrocytes do stop proliferating they synthesize Indian hedgehog (Ihh) which subsequently acts to synthesize PTHrP, creating a negative feedback loop which drives growth plate development, and chondrocyte proliferation and hypertrophy (Figure 3.) (Karimian, Chagin, and

Sävendahl 2012; Kronenberg 2003; Kronenberg 2006). (Ihh is closely related to Shh which is one of the main regulators of limb bud formation (Kronenberg 2003).) This precise process drives continued growth and development, as well as the uniform distribution of chondrocytes across the growth plate

(Kronenberg 2003; Kronenberg 2006).

Figure 3: The PTHrP-Ihh Negative Feedback Loop: Proliferating chondrocytes produce PTHrP which retards the production of Ihh (1). However, hypertrophying chondrocytes produce Ihh which increases their mitosis (2). Ihh also stimulates PTHrP production which again retards their proliferation (3). Adapted from Kronenberg 2003, 334.

Another vital part of the growth plate’s chondrocyte proliferation comes from both FGFs and

BMPs which are both active during every stage of endochondral bone formation (Karimian, Chagin, and

9 Sävendahl 2012; Kronenberg 2006). FGF signaling aids in the differentiation of hypertrophic chondrocytes, whilst BMP’s increase proliferation of chondrocytes (Kronenberg 2003). Once again, these opposing effects drive bone growth. Subsequently, Ihh expression is increased through BMP signaling feeding back into the PTHrP-Ihh loop (Karimian, Chagin, and Sävendahl 2012). Plus, FGF proteins have been tied to the growth plate senescence and thusly are responsible for the determination of adult skeletal size (Karimian, Chagin, and Sävendahl 2012).

Along with these two processes, there are a host of other factors active in the growth plate, only some which will be mentioned here. Runx2 drives the differentiation of chondrocytes from proliferative to hypertrophic forms (Kronenberg 2003). Thyroid hormones regulate chondrocyte proliferation and hypertrophy, with defects in longitudinal growth being seen in individuals with thyroid irregularities

(Karimian, Chagin, and Sävendahl 2012). C-type natriuretic peptide promotes chondrogenesis through the stimulation cell adhesion (Karimian, Chagin, and Sävendahl 2012). Vitamin D aids in the organization and maintenance of the growth plate (Karimian, Chagin, and Sävendahl 2012). Glucocorticoids, and androgens aid in shaping the growth plate, especially in defining its width (Karimian, Chagin, and

Sävendahl 2012).

During puberty the growth plates reach maturity and eventually are replaced by bone, ceasing longitudinal growth. To date there is still much unknown about the factors contributing to growth plate closure, but several factors are believed to play a part (Emons et al. 2011). Estrogen has been found to have a key effect in stimulating maturation of the growth plates, leading to fusion and the replacement of the growth plate with bone mineral (Emons et al. 2011). Another consideration is that cells within the growth plate may undergo hypoxic induced cell death, seen in in vitro testing through the upregulation of hypoxia-inducible factor 2! mRNA (Emons et al. 2011). Again, there are likely a host of factors involved in the process of growth plate closure.

As described above, a plethora of growth factors, transcription factors, and hormones are involved in growth plate formation, maintenance, and closure. These define the size and shape of bones

(Burdan et al. 2009). In other words, the growth plate is genetically programmed to grow bone to a

10 specified size and shape (Bertram and Swartz 1991; Karimian, Chagin, and Sävendahl 2012). This confirms that the skeleton is an organ of unprecedented complexity with effects of pattering and cell differentiation during development driving growth (Karsenty 2003). This is further confirmed by the coordination needed to produce perfectly formed, bilaterally symmetrical structures, a process which cannot possibly be primarily guided by mechanical forces, which in comparison are relatively simple, and unlikely to achieve these highly complex structures. The genetics of the growth plate must be the primary guidance for bone formation and development.

Proposed Research Hypothesis

The aim of this study is to show that bone development within the diaphysis is not controlled by mechanical forces. Rather its design is by means of genomic controls. It is proposed that the morphology of the bone is simply the wake of the growth plate’s temporary form and it is genomic influences which are the primary guidance (Lovejoy 2020). This hypothesis will be tested by making a cross sectional comparison of tibias from the Libben Osteological Collection. I expect that the shape of the diaphysis conforms to a standard that is dictated by the shape of the base of the physis during growth. If this is the case, it stands to reason that there are primary genetic influences on the shape which the bone takes during development.

METHODS

The Libben Osteological Collection

The Libben collection was obtained via a 1967-1968 excavation (Lovejoy et al. 1977; Mensforth

1985; Mensforth et al. 1978). Primarily a Late Woodland cemetery, it was located on the northern bank of the Portage River in Ottawa County, northern Ohio, located 6 miles upstream of Lake Erie (Lovejoy et al.

1977; Meindl, Mensforth, and Lovejoy 2007; Mensforth 1985; Mensforth et al. 1978). From the site 1327 individuals were recovered, ranging in age from 4 months in utero to +70 years (Lovejoy et al. 1977;

Mensforth 1985; Mensforth et al. 1978). Of the 1327 individuals recovered 452 are infants and children under 10 years of age (Mensforth et al. 1978). The site dates to between 800-1100 A.D. with indications it was occupied for 250-300 years (Lovejoy et al. 1977; Mensforth 1985; Mensforth et al. 1978). The population size for the site may have raged between 88 and 106 individuals at any given time (Mensforth

1985).

The ageing of the infants was conducted primarily through dental analysis, and secondarily through long bone length (Lovejoy et al. 1977; Meindl, Mensforth, and Lovejoy 2007; Mensforth et al.

1978). For adolescents epiphyseal development and closure were used to estimate age (Lovejoy et al.

1977). For the adults a selection of seven criteria was employed, with a summary age based upon a weighted mean of all available age indicators (Lovejoy et al. 1977). For those within the first year of life target ages were defined at one month intervals (Mensforth et al. 1978). For all following ages one year intervals were used based upon published trends (Mensforth et al. 1978).

With the Libben site being inhabited year round the diet indicates a hunter-gatherer/fishing lifestyle with secondary proto-agricultural practices (Kramer 2017; Meindl, Mensforth, and Lovejoy

2007; Mensforth 1985). Evidence shows a heavy reliance on freshwater fish, small mammals, and migratory birds as the key animal proteins, most likely caught through trapping (Lovejoy et al. 1977;

12 Meindl, Mensforth, and Lovejoy 2007; Mensforth 1985; Mensforth et al. 1978). It is unlikely maize was heavily cultivated, however from Kramer’s analysis of the Libben dental calculus maize was being consumed (Kramer 2017; Lovejoy et al. 1977; Meindl, Mensforth, and Lovejoy 2007; Mensforth 1985;

Mensforth et al. 1978). A variety of berries, nuts, acorns, wild rice, and millet supplement the protein of the diet (Kramer 2017; Mensforth 1985).

Several pathological conditions have been identified for the Libben individuals. There is a high frequency of porotic hyperostosis and periostitis, along with long bone growth delays (Meindl,

Mensforth, and Lovejoy 2007; Mensforth 1985; Mensforth et al. 1978; Mensforth and Lovejoy 1985).

Porotic hyperostosis is a cranial lesion usually seen with chronic hemolytic anemia, and iron-deficient anemia (Mensforth et al. 1978). Periostitis, found on the long bone shafts, manifests as layers of bone which form a ‘scab-like’ appearance over the regular cortex (Mensforth et al. 1978). The cause of these periosteal lesions is believe to be tied to chronic infection, and it is commonly seen in association with porotic hyperostosis (Meindl, Mensforth, and Lovejoy 2007; Mensforth et al. 1978; Mensforth and

Lovejoy 1985).

The porotic hyperostosis lesions are seen most frequently in the 6 to 12 month age group of

Libben children, with a lower frequency of occurrence from 1 to 3 years (Mensforth et al. 1978). After the

3 years the condition declines and remains infrequent throughout the remainder of childhood (Mensforth et al. 1978). The levels of the lesions themselves are also fairly low, indicating only mild iron-deficiency anemia (Mensforth et al. 1978). The majority of unremodeled periostitis lesions are seen in the first year of life, with the frequency declining quickly from 1 to 3 years of age (Mensforth et al. 1978). Periostitis is commonly found in association with acute infection, and studies have shown high levels of “pneumonia, septicemia, otitis media, staph infections, and gastroenteritis among infants and young children during the first year of life in preindustrial societies,” indicating this to be the likely cause within the Libben subadults (Mensforth et al. 1978).

Infection being identified as the cause of the periosteal lesion also gives cause to the degree of long bone growth delay seen within the population (Lovejoy, Russell, and Harrison 1990; Mensforth

13 1985; Mensforth et al. 1978). Iron deficiency anemia can also be considered part of the cause of growth delay in the subadults (Mensforth 1985). These growth delays show a one year ‘lag’ in development within the first five years of life, with the most distinct period of delay being between 6 months to 2 years

(Lovejoy, Russell, and Harrison 1990; Mensforth 1985; Mensforth et al. 1978). Despite this impedance, growth rates appear to recover after 5 years of age, with a distinct period of ‘catch-up’ growth between

8.5 and 11 years of age (Lovejoy, Russell, and Harrison 1990; Mensforth 1985; Mensforth et al. 1978).

By 10 years of age 70.5% of the eventual adult tibia diaphyseal length was achieved (Mensforth 1985).

Despite these pathologies and the significant growth delay in the Libben subadults it is not anticipated that this will influence the results of the project. As this will be a cross-sectional study of solely the Libben collection any abnormalities should be consistent across the sample. As only the exterior shape of the diaphysis in correspondence to the shape of the growth plate base is being considered, any growth delay in length should not be of concern for the results. The growth plate perimeter shape can be discerned within the diaphyseal circumference regardless of these conditions.

Sample Selection

From the full collection of 1327 individuals, an initial 183 tibias from 130 individuals, aged between fetal to 19 years were selected based upon a variety of criteria. Tibias were chosen as the focus of the study due to their role as the most weight bearing long bone in the body. Also, in the above cited literature where experimentation has been conducted the tibia has often been the site of study due to assumptions that it would receive the greatest loading forces and therefore show the most effect from stress and strain. In keeping with prior studies, the tibia was selected to corroborate results.

The 183 individual tibias were initially selected based upon their condition of preservation.

Specifically, it was required that the proximal and distal physes were intact, and the whole length of the diaphysis was preserved. From those 183 a subset of 36 tibias was selected from 30 individuals. This subset was selected based upon a higher degree of preservation. The proximal and distal growth plates had to be in a condition which preserved the entire perimeter shape, and the shaft of the bone had to have no more than one clean, reparable break, to reduce the possibility of deformation to the diaphyseal

14 perimeter shape. If fusion of the growth plates had already occurred, the line of recent fusion had to be clearly recognizable.

The 30 individuals initially selected ranged in age from an infant under 1 year, to a 17-year-old male, with recent fusion of the growth plates. Of these 30, the largest, belonging to the 17-year-old male, was selected for further analysis. This was specimen #02062, a left tibia, with recent fusion of both the proximal and distal epiphyses. Length was estimated from distal line of recent fusion to proximal line of recent fusion through an average of four measurements: the most extreme anterior, posterior, medial, and lateral lengths. This provided an estimated length of 32.1cm for this specimen. Although the focus of the study remained on specimen #02062, a total of 34 individual tibias were three dimensionally scanned to create computer generated models for future study.

Three-Dimensional Scanning and Printing

An “Artec Eva structured light handheld 3D scanner” was used to capture accurate, textured images of the tibias. It provided high resolution, precisely measured images, with an accuracy up to

0.1mm, and a resolution up to 0.2mm (“3D Object Scanner Artec Eva | Best Structured-Light 3D

Scanning Device” n.d.). Each tibia required two scans to be taken to capture the full surface, one with the bone laid on its posterior surface, and the second with it laid on its anterior surface.

The scans were input into Artec Studio 15, a software package for processing 3D scanning data.

Once both surface scans had been captured several steps were required to clean the scans and align them to create the final three-dimensional model. The first step was to run an HD Reconstruction, a preset tool within Artec Studio 15, which ensured the highest quality output. From there, all unwanted scanned data had to be erased. This removes all unwanted visual noise picked up during the scanning process, as well as the surface on which the tibias were set to conduct the scans. Two settings were used to ensure all unwanted material was erased, the ‘cutoff plane selection’ tool which aids in removing the scanning surface, and the ‘2D selection’ which allows for a greater manual control over individual pixel erasure.

Once all unwanted data were removed the two scans were then aligned using a set of three selected points. Each bone required different points to be chosen dependent on the unique features of the

15 tibia itself, but this enabled the software to ensure the two scans attached to one another accurately. Once the ‘align’ function was executed the two scans come together as a single whole model of the original tibia.

The remaining process involved cleaning the model using the ‘outlier removal’, ‘sharp function’,

‘small object filter’, and ‘mesh simplification’ tools, all presets within the Artec Studio 15 program designed to provide a final high precision model of the scanned object. Final maximum error within each scan was 0.1%, estimated by the software package. This was seen as acceptable as all scans and processing were conducted by the same individual, with the same scanner and software package.

Figure 4: 3D Scans of Specimen 10063: #10063 was the smallest tibia able to be scanned, from a 3-year-old individual, measuring 15.1cm. As can be seen in the lower two images, resolution began to decrease at this size. This meant that the lowest limit in size for scans was set at this point. Top: the diaphysis positioned with the proximal surface to the left; Bottom Left: the proximal growth plate surface; Bottom Right: the distal growth plate surface.

Figure 5: 3D Scans of Specimen 03210: #03210 is a right tibia from a 13-year-old individual, measuring 21.5cm in length. Top: The diaphysis positioned with the proximal surface to the left; Bottom Left: The proximal growth plate surface; Bottom Right: The distal growth plate surface.

Figure 6: 3D Scan of Specimen 02062: #02062 is the largest tibia in the sample, a left tibia from a 17-year-old male. Image shows the full diaphysis with both proximal (upper right) and distal (lower left) epiphyses fused to the shaft. Lines of recent fusion can be discerned on both the proximal and distal ends of the diaphysis.

17 Once the 3D scan of specimen #02062 had been collected and processed the next step was to create a 3D printing of the model. The purpose of creating a 3D replica of the tibia allowed for cuts to be made, cross sectioning the bone, without damaging the original specimen and allowing for the final analysis to be conducted. The 3D scan of this tibia was printed using a Stratasys FDM F370 series 3D printer, with an accuracy of +/- .200mm (“Stratasys F123 Series 3D Printers” n.d.). It was printed using polylactic acid (PLA), a form of thermoplastic commonly used in 3D printing.

Figure 7: Comparison of Specimen #02062 and the 3D Printed Replica: Left: Anterior Surfaces (left specimen #02062, right replica); Right: Posterior Surfaces (left specimen #02062, right replica).

Final Processing for Analysis

The final steps in the process were to cross section the printed tibia, capture images of younger bones to be used for comparison and create overlays to assess the similarity between the cross sections and the original growth plate surfaces. It was assumed that each cross section should correspond to a tibia which represented specimen #02062 at the moment of development during which it measured an assigned length. As growth proceeds approximately twice the rate below the nutrient foramen as above, focus was directed on just the proximal third of the bone, the 10.37cm from the nutrient foramen to the proximal line

18 of recent fusion. The 10.37cm distance was divided equally, and four cuts were made at 3.07cm, 5.15cm,

7.22cm, and 9.30cm from the nutrient foramen, working toward the proximal end.

Figure 8: Cross-Sectioning of the 3D Printout: The nutrient foramen was marked at 10.37cm from the proximal line of recent fusion, and four cuts were made, cross-sectioning the body of the diaphysis.

Based upon these cutting distances four specimens were selected for comparison from the initial subset of 36: specimen #12206, 7.56cm in length, from an infant under 1 year of age; specimen #3203,

14.0cm in length, from a 3-year-old individual; specimen #5035, 19.9cm in length, from a 6-year-old individual; and specimen #4055, 25.9cm in length, and from a 13-year-old individual.

Figure 9: Specimen #02062 Alongside Comparison Specimens: (From top to bottom) Specimen #12206, Specimen #3203, Specimen #5035, Specimen #4055, and Specimen #12206, aligned with proximal surfaces to right.

19 The four selected specimens were then photographed using a “16-megapixel Panasonic Lumix

DMC-FH25” camera. Images were captured of both the proximal and distal growth plate surfaces by setting the bones in a sand tray to hold them steady whilst photographs were taken. Then the cross sectioned surfaces of the 3D printed model were also photographed, using the same camera.

Final comparison of the growth plate surfaces to the cross-sections of the 3D printed tibia were done using the software GNU Image Manipulation Program, version 2.10. First each photograph was labeled with lines indicating the anterior-posterior and mediolateral axis. Next the labeled image of the cross section was loaded as a layer, with the corresponding labeled photograph of the tibia growth plate loaded as a secondary layer. The transparency of this secondary layer was reduced until both images could be clearly seen, one on top the other, and finally they were aligned based upon the center point of the anterior-posterior and medial-lateral labeling.

RESULTS

Image Overlays

Following the comparisons and subsequent layering of the cross-section images with the photographs of the proximal and distal growth plate surfaces it became clear that the anticipated result was not obtained.

Figure 10: Side-by-Side Comparisons Specimen #12206 and Slice 1: Slice 1 was taken 3.07cm proximal from the nutrient foramen. Left: Proximal Growth Plate of specimen #12206 with anterior-posterior (left-right) and medial- lateral (top-bottom) axis marked; Center: Slice 1 of 3D printed replica of specimen #02062 with anterior-posterior and medial-lateral axis marked; Right: Distal Growth Plate of specimen #12206 with anterior-posterior and medial- lateral axis marked

Figure 11: Overlay Comparisons of Slice 1 and Specimen #12206: Slice 1 was taken 3.07cm proximal from the nutrient foramen. Left: Proximal growth plate surface of #12206; Right: Distal growth plate surface of #12206.

Figure 12: Overlay Comparisons of Slice 2 and Specimen #3203: Slice 2 was taken 5.15cm proximal from the nutrient foramen. Left: Proximal growth plate surface of #3203; Right: Distal growth plate surface of #3203.

Figure 13: Overlay Comparisons of Slice 3 and Specimen #5035: Slice 3 was taken 7.22cm proximal from the nutrient foramen. Left: Proximal growth plate surface of #5035; Right: Distal growth plate surface of #5035.

Figure 14: Overlay Comparisons of Slice 4 and Specimen #4055: Slice 4 was taken 9.30cm proximal from the nutrient foramen. Left: Proximal growth plate surface of #4055; Right: Distal growth plate surface of #4055.

Figure 15: Overlays of Proximal Growth Plate Surfaces: Proximal growth plate surface of Specimens #12206, #3203, #5035, and #4055

As soon as a simple visual comparison was made it became clear that the growth plates and the perimeter shape of the mature tibia’s diaphysis do not align with one another. It was anticipated that the growth plate shape of each specific specimen would directly resemble the perimeter of the diaphysis at the point where each corresponding cut was made, and that further analysis would yield a numerical percentage of similarity or difference. It is clear however that no further analysis is needed at this time as the difference between the growth plates and the slices is so extreme.

From the overlays it is possible to see more clearly where the differences lie and that at no point does the perimeter shape of the growth plate correspond with the perimeter shape of the cross sectioned tibia. The proximal growth plate consistently shows a greater medial-lateral width, whereas the adult

23 tibia’s diaphysis shape has shifted so the anterior-posterior axis is greater, regardless of which specimen is used for comparison. The extent of the medial-lateral consistency between the specimen growth plates can be seen in figure 15, where the four comparison specimens’ proximal growth plate surfaces are overlaid for contrast. Similarly, the distal growth plate’s shape is consistently different from that of the mature bone. However, in the case of the distal growth plate it does conform to the maximum exterior dimensions of the adult tibia medial-laterally, so that it sits within the perimeter contour of the bone. This suggests that as growth moves posteriorly considerable modification is taking place after the advancement of the growth plate, whereas distally additional bone is being laid down, especially along the anterior- posterior axis, but very little is being removed following advancement.

CONCLUSION

The results of the image overlays fail to support the hypothesis that bone growth develops as the wake of the growth plate and will be observable within comparisons of cross-sections of a mature diaphysis to growth plate surfaces of juveniles, as defined by their lines of fusion between the diaphysis and epiphysis. There is no pattern observable within the images to support this form of the hypothesis.

From the proximal end to midshaft the long axis shape changes dramatically from widest mediolaterally to anteroposteriorly. Mediolateral dominance is clearly necessary for normal knee function and to accommodate the femoral condyles, which suggests that the distinct shift in long axis dominance to anteroposterior must reflect some yet unknown genetic process. This unknown process will surely have a far-reaching history, given that the tibia is recognizably similar in almost all mammals, suggesting this must be the primary area of hox function.

Interestingly, when right left comparisons are made of tibias from the same individual, the similarity between them suggests that the growth plate must be under extensive genetic control to produce two exactingly similar but mirror-imaged cross sections. This suggests that genetically directed remodeling must be incredibly conserved within the genome for this degree of right left symmetry to be so consistent, despite the massive number of genomic changes which are clearly present during growth, as demonstrated from the dramatic changes found in the overlay findings.

Although further analysis was not required in this case, we proposed that future studies be conducted to trace the development of the diaphyseal shape during growth. One option may be to CT scan directly below the growth plate, capturing thin slices to be able to pinpoint the transition of long axis dominance. Another option would be to make comparisons directly on an individual through time, rather than in a cross-comparative manner. This could be done using rodent models and CT scans, capturing images throughout the developmental period, and conducting longitudinal comparisons of the growth

25 plate to the diaphysis. This may give explanation to how and when the long axis of growth changes so distinctly.

With the significant long axis shift from medial-lateral to antero-posterior dominance, it is proposed that there is much yet to be learned about the region of bone growth and development. It is suggested that rather than a thin growth plate, there is an area of growth which comprises both the joint surface, growth plate, and the region where the long axis shift is occurring. This ‘growth area’ is likely maintained by a hox program and subjected to cartilage modeling. This would mean that there is not a simple line of fusion between the joint complex and shaft, but rather a substantial region of growth.

Whatever the future direction of study, there are gaps in the current literature, and a full understanding of how the growth plate, or growth area, is gnomically controlled and subsequently patterns growth has yet to be ascertained. The degree of right-left similarity, and the high degree of anteroposterior long-axis shift during growth has not been adequately explained by Wolffian hypotheses.

Expanding research into the genomics of growth plate shape, providing a greater knowledge of how bone shape is created, what genetic controls are regulating and patterning growth, and how bones achieve their final form, will allow for a fuller understanding of the relationships between growth, development, and maturation. This in turn will have an impact on our understanding of the relationships between bone and soft tissue, gait, and most importantly natural selection, given that genetics is the root of the process.

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