How the Immune System Determines Bone Graft Success or Failure:

A histological analysis of rabbit tibia and human extraction sockets

The immune system plays a critical role in determining the success or failure of bone regeneration. Trauma, infection, thermal, or chemical injury results in an immediate, non-antigen specific immune response. This response is controlled by cells of the innate immune system and is called acute inflammation. Acute inflammation is the first stage of healing and is intended to remove the cause of the injury, remove any damaged tissue, and set the stage for the regenerative process to restore normal form and function.

The process of acute inflammation is initiated by resident immune cells already present in the involved tissue, mainly resident macrophages, dendritic cells, histiocytes, Kupffer cells, and mast cells. These cells possess surface receptors known as pattern recognition receptors, which recognize two sub-classes of molecules: pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs are compounds that are associated with various pathogens, but which are distinguishable from host molecules. DAMPs are compounds that are associated with host-related injury and cell damage. The mediator molecules also alter the blood vessels to permit the migration of leukocytes, mainly neutrophils, and macrophages, outside of the blood vessels into the tissue.

Acute inflammation requires the production of pro-inflammatory substances such as cytokines. These may include tumor necrosis factor-alpha (TNFα), Interleukin 1 beta (IL1β), IL-6, IL-8, and chemokines such as macrophage chemotactic protein 1 (MCP-1) and reactive oxygen intermediates (iNOS).

Together these cells and molecules eradicate microbes or toxins, remove damaged tissue, and recruit more cells to begin the regenerative phase. The acute inflammatory phase is guided by M1 macrophages of the innate immune system.

When the acute inflammatory response has removed the offending agent and cleared the damaged tissue, the pro-inflammatory M1 macrophages are polarized into anti-inflammatory M2 macrophages. M2 macrophages produce Interleukin 10 (IL-10), IL-1 receptor antagonist (IL-1ra), and Transforming Growth Factor beta (TGFβ), and subsequently direct regeneration that restores normal form and function. However, if acute inflammation is not able to eradicate the cause of the injury in a timely manner, chronic inflammation develops and regeneration is blocked. In chronic inflammation, the primary immune cells are M1 macrophages and T lymphocytes, which produce cytokines and enzymes that cause more lasting damage to local cells and tissues. In chronic inflammation, resolution of the acute infection gives way to ongoing tissue damage and destruction, manifesting as tissue fibrosis in soft tissue and sclerosis in bone.

With this knowledge we can discuss and understand why some bone grafting materials produce regeneration and why other bone grafting material produces fibrosis and sclerosis.

In histologic studies of bone, virtually all bone samples are taken with a trephine prior to implant placement. Dentistry makes the assumption that after loading, the bone graft particles will be resorbed and mature lamellar bone will support the implant. However, dentistry has no scientific support for this assumption and not knowing what type of bone is produced after loading can make the difference between implant success or implant loss. In order to ascertain the type of bone that forms under load, we studied bone histology in a rabbit tibia model. Bone grafts placed into rabbit tibia will gradually become loaded as the bone grows and the animal puts load while walking and supporting itself.

New Zealand White Rabbit

Left tibia is prepared with two osteotomies. The top is grafted with Socket Graft and the bottom with Socket Graft Plus.

New Zealand White Rabbit

Right tibia is prepared with two osteotomies. The top is grafted with Sinus Graft, and the bottom with allograft.

Socket Graft at 6 weeks. Woven bone is being remodeled by basic multicellular units into lamellar bone. The acute inflammation has resolved and no chronic inflammation is present. Conversion from woven bone into lamellar bone indicates M1 macrophages have polarized into M2 macrophages and the process of regeneration is occurring.

High power from the previous histology at 6 weeks showing a basic multicellular unit in the process of converting woven bone into lamellar bone. Osteoclasts are resorbing bone on one side and osteoblasts are forming lamellar bone on the opposite side. Note the relative size and shape of the cells.
Low power 8 weeks after grafting with Socket Graft. The regenerative process is complete and the osteotomy is filled with mature cortical bone.

Socket Graft, when grafted into the tibia of New Zealand White rabbits, demonstrates the conversion of acute inflammation caused by the surgical intervention by polarizing M1 macrophages into M2 macrophages, which converts the acute inflammatory phase into regeneration. Within an 8-week time period, the osteotomy is completely regenerated.

Next, we see the histology at the same time frames for mineralized freeze-dried bone allograft. The process of allograft mineralization has never been published in medical or dental literature, so enjoy seeing this process for the first time.
Mineralized freeze-dried bone allograft at 6 weeks, low power. At this time, the histology shows closely packed allograft particles surrounded by chronic inflammation.
Mid-powder of the histology at 6 weeks shows initial mineralization and intense chronic inflammation predominated by lymphocytes of the acquired immune system. Note the separation artifacts between the allograft particle and the newly formed mineralized tissue.
Low power at 8 weeks. This tissue shows a reduction in the percentage of graft particles remaining in the tissue. In addition, there is an increase in the amount of bone covering the allograft particles and a reduction in the amount of chronic inflammatory infiltrate.
A higher magnification at 8 weeks shows osteoclast resorption of the cadaver bone graft particle. The osteoclasts are abnormally large, unlike a normal osteoclast. The osteoclast located in the center of the slide is the largest cell we have seen throughout the years of viewing bone histology. This is the first time an osteoclast has been found on the surface of an allograft particle. This confirms that osteoclasts are capable of resorbing mineralized allograft particles. Note the separation artifact between the newly formed bone and the allograft particle.
At eight weeks, the lymphocyte infiltration remains in localized areas, but is overall reducing. Note the separation artifact between the newly formed bone and the allograft particle.

Chronic inflammation is reduced at 8 weeks, but still significant. SteinerBio is the first to show allografts being resorbed by osteoclasts. However, only a small amount of mineralization has occurred and the process of mineralization and resorption is obviously dysfunctional, showing abnormal cells and abnormal mineralization described as sclerotic bone.

The following histology is a site grafted with our bone graft putty mixed with our βTCP particles. This will allow for a direct comparison between resorption and mineralization of our biocompatible bone grafts and cadaver bone grafts.

Low power of a rabbit tibia grafted with Immediate Graft, which is our putty mixed with βTCP particles. The histology shows osteoconduction with bone-forming throughout the majority of the site and without inflammation.

Mid power of the previous histology slide. This shows bone formation over the particles which is a process called osteoconduction. All particles are in contact with a basic multicellular unit composed of osteoclasts on the surface of the βTCP particle and osteoblasts forming lamellar bone. No inflammatory infiltrate is present.

This study outlines the difference between bone regeneration that occurs when SteinerBio bone grafts are grafted into New Zealand White rabbits and the sclerotic bone that is produced by cadaver bone grafts.

But what about humans?

Again, there is no published histology of cadaver bone grafts in humans in the process of mineralization. The following histologic samples will compare SteinerBio bone grafts to mineralized free-dried bone allograft at similar time frames in human extraction sockets.

Extraction socket in a human at 6 weeks. The site shows approximately 40% mineralization with robust osteoblasts producing woven bone. No inflammation is present.
Higher magnification shows robust osteoid formation lined by osteoblasts. Osteomacs are M2 macrophages that guide bone regeneration. No inflammation is noted.

This histology is a low power of a site grafted with Socket Graft after 6 months. The osteoblasts produce mineralized tissue until there is no space left except blood vessels. The mineralization shown in this histology is much greater than normal bone production and when normal bone is formed beyond normal, the process is defined as stimulating osteogenesis. A high percent of mineralized tissue is good only if that tissue is capable of converting into normal cancellous or cortical bone.

This histology is from a site grafted with Socket Graft 6 months after loading. When SteinerBio products are loaded, they remodel and form lamellar bone ideal for carrying load. This image documents the conversion of immature woven bone to mature lamellar bone ideal for carrying load and completing the regenerative process.

This histology is from a human extraction socket grafted with mineralized freeze-dried bone allograft at 7 weeks. The cadaver graft is labeled and the extent of the new mineralization is noted. The site is filled with chronic inflammatory cells that have been identified as cytotoxic T cells, also called Killer T cells. These cells are responsible for organ rejection.
This is a core sample taken from a human socket 6 months after grafting with mineralized freeze-dried bone allograft. There are no osteoclasts present and the cadaver particles are covered with mineralization. When the cadaver bone graft particles become covered with mineralization, chronic inflammation subsides and the particles remain encased in what appears to be woven bone. However, because there are no basic multicellular units, this bone never remodels into lamellar bone and therefore is termed sclerotic bone. Allografts have never been shown produce cancellous or cortical bone.
This histology is from a site grafted 15 years prior with a combination of mineralized freeze-dried bone allograft and Bio-Oss. The bone was loaded with an implant for 4 years prior to the implant failing. Allografts and Bio-Oss form sclerotic bone and never are resorbed or “turn over” into normal bone. In this histology and all allograft histology, normal cancellous or cortical bone is never produced and the bone remains sclerotic.

The human and animal histology of cadaver bone grafts in the process of mineralization has never been published, so you are seeing this for the first time. No xenograft or allograft is ever fully resorbed. The chronic inflammation associated with cadaver bone grafts produces sclerotic bone that covers the cadaver bone particles and isolates the particles from the patient’s immune system. We have shown that cadaver bone graft particles are capable of being resorbed, however, the chronic inflammation produces mineralized tissue that never resorbs. We postulate that the purpose of this mineralized tissue is to protect the host from the inflammatory response caused by the graft material.

After a site is grafted with cadaver particles, the socket is initially filled with approximately 90% cadaver bone graft particles, but ultimate resorption stops at approximately 30%. All studies have shown that sites grafted with cadaver bone graft particles remain at approximately 30% with no change over time. A scar on the surface of the skin is fibrosis caused by the development of chronic inflammation and is permanent. Likewise, chronic inflammation in bone produces fibrosis that mineralized and becomes permanent sclerotic bone. The scar tissue formed in sockets grafted with cadaver bone grafts has been shown to produce marginal bone loss in a number of studies and in the future, the dental profession will find that the primary reason for implant loss will be placing implants in sockets grafted with cadaver bone grafts, which is currently misdiagnosed as peri-implantitis.

The dental profession refers to guided bone regeneration (GBR) when using cadaver bone grafts. Cadaver bone grafts never regenerate normal bone and using the term “regeneration” in association with cadaver bone grafts is inaccurate.

The dogma associated with cadaver bone grafts has no scientific support and it is time for the dental profession to move on to the science of regenerative medicine.


American Society for Bone and Mineral Research (ASBMR)

Tissue Engineering and Regenerative Medicine International Society (TERMIS)