The concavity: The ‘shape of life’ and the control of bone differentiation

South African scientists, in collaboration with their international counterparts have made ground-breaking discoveries in the field of bone research. How does bone regrow? Can we initiate bone regeneration? What are the triggers and what does shape have to do with it? In this article the inspirational Professor Ugo Ripamonti, Director of the acclaimed Medical Research Council funded Bone Research Laboratory, based at the University of the Witwatersrand takes a personal voyage charting his entry into the world of scientific enquiry, detailing the role of his mentor Prof Hari Reddi in championing a career spanning countries and decades in search of the answers to questions formulated many years ago. The article below is followed by a full scientific paper which details the scientific research that has now led to a wealth of opportunities yet to be explored in turning these novel findings into a reality for everyday South Africans.

Ugo Ripamonti

Director, Bone Research Laboratory, a Research Unit of the South African Medical Research Council and the University of the Witwatersrand, Johannesburg

How did I become interested in geometry? It all started many years ago when visiting the laboratories of Hari A. Reddi PhD, then Chief of the Bone Cell Biology Section at the National Institutes of Health, Bethesda, Maryland, USA. We met, Hari and I, in 1986 and since then we have grown into a rare scientific friendship that often binds scientists across continents and cultures, and initiates novel opportunities to contribute to a progressive understanding of Nature. After meeting Hari, and working with his staff at the then Bone Cell Biology Section, I learnt the scientific discipline needed to record the observations of the biological universe, becoming entranced by the fascinating phenomenon of “Bone: formation by autoinduction” to which Dr Reddi had made substantial contributions, mentoring many students across continents.

Over decades of interest in bone cell biology, no single concept has captured my imagination to a greater degree than the phenomenon of the induction of bone formation. How do new bones grow? My studies over the past several years were also the result of a passion for this fascinating aspect of bone cell biology. As Arthur Kornberg so clearly states in his Science Editorial, the “ultimate scientific languages used to report results are international, tolerate no dialects, and remain valid for all of time”. I have also learned that “the pursuit of the free spirit of enquiry” is the most stable and powerful of human endeavours, and is continuously helping to reveal the extraordinary intricacies and awesome beauties of Nature (Kornberg 1992).

As any biological scientist knows, many long nights and hours of dedication are needed, performing many experiments, isolating and purifying proteins. When I reflect on working in the eighties at the Bone Cell Biology Section, Bethesda, the amount of work has been simply staggering, learning procedures and experiments, chromatography and purification of proteins such as osteogenin from baboon bone, working late into the night running techniques used by many scientists in the field such as adsorption and affinity chromatography purification columns. Osteogenin was important in the study of bone growth. It is a protein that initiates bone formation, its name being derived from osteo meaning bone and genin meaning origin. This protein is part of a family of proteins called the bone morphogenetic proteins (BMPs) known to be powerful initiators of the induction of bone formation.

The exoskeletons of certain sea corals have cancellous pore structures similar to bone. The original structure of the calcium carbonate [CaCO3] exoskeleton can be preserved during a process known as hydrothermal conversion at high temperatures and pressures to hydroxyapatite [Ca10(PO4)6(OH)2; HA] which is the mineral found in bone and teeth. My earlier doctoral studies at the Dental Research Institute at the MRC/University of the Witwatersrand, Johannesburg under the guidance of Prof PE Cleaton-Jones, identified that the hydroxyapatite from coral provides a porous support which is an ideal material for bone in-growth or osteoconduction from the viable interfaces of the severed margins of bone. More importantly, however, I have also learned that these coral-derived porous constructs also had the unique capacity to spontaneously and intrinsically initiate the induction of bone formation - that is, to make new bone even when implanted in non-skeletal intramuscular sites where there is no bone, in primates. More importantly, bone can form without the need for application of bone inducing proteins such as osteogenin, within these materials which mimic bone structure.

In Bethesda, the desire to use the calcium carbonate exoskeleton of the sea-coral as a porous construct or vehicle to deliver the biological activity of the bone formation-inducing (bone morphogenetic) proteins extracted and purified from baboon bones, was overwhelming.

Again, purifying the proteins was a critical factor in investigating this further. I was then helped by a dedicated student, Laura Yeates, who significantly contributed to the study, purifying the baboon osteogenin protein, greater than 50-000 fold from crude extracts. Laura contributed to other unique topics of research of the laboratory, further purification of baboon osteogenin for chromatographic preparation of macroporous matrices to present bone inductive proteins at in vivo sites, and additional studies on the ‘spontaneous’ induction of bone formation in structures that mimic the pores and geometry of bone. In the early nineties, her purification of baboon osteogenin, was simply faultless. Working often late in the laboratories of the Bone Cell Biology section, Laura further purified another batch of baboon bone matrix and managed to isolate proteins with substantial osteogenic activity which were then adsorbed onto the coral-derived constructs as a novel delivery system for bone-inducing proteins. Further contributions were also shown by Laura’s desire to run in vitro studies with osteoblast-like cells (cells derived from bone) to be combined with small discs of coral-derived macroporous constructs to form a bone bioreactor in vivo for potential transplantation.

The study, set to provide a structure that contains living cells, or a bioreactor, to form new tissue when implanted in non-bony sites in rodents, has never been published nor the fascinating observation of cell proliferation within the pores of the coral-derived structure when evaluated in the laboratory. The preparation of this Science in Africa manuscript on the effect of the geometry on the induction of bone formation has created the opportunity to present such digital images on the effect of shape or geometry of the bone-mimicking structures to align cells in such a way that they differentiate to make bone.

At the Dental Research Institute and then to the NIH, flying frequently to the US, Bethesda, I shared the dream of using coral-derived porous hydroxyapatite structures to deliver the biological activity of the naturally-derived highly purified osteogenin. I thus spoke to Shu-Shan Ma, a medical doctor from China and a research associate in the laboratories of Dr Reddi, who said: “you shall need to speak to the Chief; Dr Reddi is not particularly keen to use any mineralized matrix in his subcutaneous assay in rats with osteogenin”. I spoke to Dr Reddi nevertheless, and he said: “No, it is of no use, a waste of rats but above all of osteogenin”. He also said: “Remember, to obtain the induction of bone formation, the mineralized bone matrix is firstly demineralized; the mineral phase inhibits the differentiation of bone”. I said then that the coral-derived hydroxyapatites were uniquely capable of inducing bone formation on their own when implanted in the abdominal muscles of the baboon - where there is no bone. Abruptly in his office (a very small office packed with books and journal papers) he brusquely said: “show me” and returned his eyes to a drafted manuscript. I then showed him the sections I was carrying with me from the Dental Research Institute in South Africa to try to understand why bone would form within the macroporous spaces without the addition of soluble proteins that would initiate the induction of bone formation.

In total silence Hari saw the sections and then called Dr Ma and said: “few rats, low doses of osteogenin, short time periods”. We then prepared the samples by reconstituting small discs of coral-derived hydroxyapatites with highly purified osteogenin from bovine bones.

Dr Reddi implanted the specimens under the skin (subcutaneous) of the animal models; the subcutaneous and/or intramuscular implantation of putative inductive substances – that is, proteins and/or matrices where there is no bone – is the acid test to unequivocally prove the capacity of the implanted proteins and/or matrices to induce new bone where there is none, i.e. “bone: formation by autoinduction”. The above defines the bona fide inductive activity of a protein and/or a device as osteoinductive – able to induce bone. The device, to be called osteoinductive, must initiate de novo bone formation in extraskeletal tissues where there is no bone.  

Implanted specimens had to be harvested on day 7 and 11 after implantation under the skin. We later carried out the required biochemical analyses, before the selection of the material for histological analyses. Dr Ma went to Dr Reddi’s office and, though he was not asked to proffer his opinion, stated: “Chief, I believe the specimens are positive” with a hint of a smile on his face. Dr Reddi stonily said: “cannot be”; Dr Ma still smiling said: “Chief, the alkaline phosphatase activity is positive, the samples are hot”. Dr Reddi without turning and rather bluntly stated – as to conclude any further discussion - “the assay is seemingly positive because samples are loaded with polymorphonuclear leucocytes as a foreign body reaction”. Dr Ma then indicated to me that we had to leave and muttered something, and we selected the tissues for microscopic analyses (histological material) for undecalcified processing.

Yet, during the finalization of the alkaline phosphatase assays, we found to our surprise that whilst blocks of macroporous hydroxyapatite with osteogenin were all positive, the particulate hydroxyapatite, i.e. identical hydroxyapatite but in granular configuration, were consistently negative. A few days later, the histological material was ready and, as predicted by the biochemical analyses, the histological sections showed the induction of bone formation by osteogenin, a bone morphogenetic protein, when reconstituted with macroporous blocks of coral-derived hydroxyapatites.

The surprises, however, were not over since the particulate granular hydroxyapatites did not show any bone formation even if pre-treated with doses of highly purified naturally-derived bone morphogenetic proteins. Dr Reddi, after my summary/presentation, said rather abruptly: “Think about it more; think and come back when you are ready and talk with me”. I then drafted a complete manuscript stating that naturally-derived highly purified BMPs/OPs when reconstituted with coral-derived macroporous constructs induce bone formation via a cartilage phase (endochondral bone), when implanted in the subcutaneous space of the rat where there is no bone. The same macroporous hydroxyapatites not in cylindrical form but in granular particulate configurations did not induce bone formation when combined with highly purified BMPs/OPs. The study has shown that highly purified osteogenin could be delivered by a material other than the insoluble collagenous bone matrix, the most commonly used carrier to reconstitute bone inducing proteins.

What is it that is different between the implants? It is the same material’s composition, isn’t it? “Oh yes, the same material, surface characteristics, the same proteins added”; and so what is it that makes such a dramatic difference? What it is that can overrule the morphogenetic power of a bone morphogenetic protein?” I then said rather timidly: “what about the shape?” Hari Reddi then smiled but severely almost taking pleasure in conducting me through the frontiers of the unknown, but hard in his mentoring, he almost shouted: “say another word, another word, say it”. “Geometry” I proffered, confused yet suddenly certain that the word had entered so prominently in my scientific career since. “Yes,” he then shouted “geometry; write a paper and this is the title: The effect of geometry of porous hydroxyapatite on the induction of bone formation by osteogenin, a bone morphogenetic protein”.

I was then drafting the paper when Charles Huggins stopped me in the corridor going to the Bone Cell Biology Section. Dr Huggins, mentor of Dr Reddi and Nobel Laureate in Medicine and Physiology, was visiting the NIH and the Bone Cell Biology Section. With a smiling face he asked: “Doctor, Doctor Reddi has told me of your very interesting results on the geometry of the various hydroxyapatite substrata you have implanted with osteogenin; tell me now, where are you planning to submit the manuscript?” I replied that we thought to submit to Matrix Biology since the Journal deals prominently with the study of extracellular matrices. Charles Huggins then smiled, and said: “Doctor, do you wish to make a ‘bang’ in life or not?” For seconds I was almost lost though suddenly the meaning of his words were very clear. Dr Huggins continued: but if you wish to make a ‘bang’, you need to publish your thoughts, ideas and creativity in Science – there is no other Journal but Science to make a ‘bang’ in your scientific career”.

And so we did, Dr Reddi rapidly correcting many manuscript’ drafts, and a few days later Dr Reddi and I dropped the manuscript with a suggested cover page to the Editorial Office in H Street Washington DC, walking into the Editor’s office. Frank Luyten, then research associate at the Bone Cell Biology Section, was somehow mesmerized (and unnerved) by the fact that weeks were passing without hearing from Science, implying thus that the manuscript has been sent to reviewers, by its self already an achievement. Science did not take the manuscript but the paper was published by Matrix Biology in 1992 with a series of colour microphotographs which highlighted that the expression of the osteogenic phenotype is regulated by the geometry of the substratum. The paper, which shortly followed my first papers on the inherent bone inductive ability of the coral-derived porous constructs was the beginning of a long series of papers on the effect of the geometry on the expression of the osteogenic phenotype. This culminated in the identification of a specific geometric configuration, the concavity: the shape of life, that resulted in the publication of important papers let alone the granting of PCT and US patents on the effect of the geometry on the initiation of bone formation.

An important goal of the Bone Research Laboratory, a Research Unit of the South African Medical Research Council and the University of the Witwatersrand, Johannesburg, Faculty of Health Sciences, has been the search for calcium phosphate-based porous carriers that by virtue of their shape would initiate the induction of bone formation even without the additional application soluble bone inductive proteins. The desire to construct porous scaffolds from calcium phosphate powder, as opposed to the use of coral-derived constructs prepared in the US, forged a tight collaborative research with the Council for Scientific and Industrial Research (CSIR), Pretoria; this collaborative effort resulted in a complex series of scientific outputs on South African calcium phosphate-based bioceramics jointly prepared and evaluated with the ultimate goal to translate research experiments in pre-clinical settings on the “geometric induction of bone formation” into clinical contexts for the benefit of the people of South Africa.

At a first glance, it is hard to understand how the geometry, which is the shape or form of a biomaterial device that is implanted into animal tissues, can so dramatically induce and control the expression of specific genes and even initiate the induction of tissue formation, in context, the induction of bone. The full feature paper in this issue of Science in Africa will try to convey to the readers the critical role of the geometric configuration of various biomaterial matrices on the induction of bone formation. The full paper detailing these findings is available here. (Note some images may be of a graphic nature).