Histological and MicroCT Analysis No Graft, Allograft, and SteinerBio Bone Grafts

A critical size defect study utilizing both histological and micro-CT analysis.

Both histological analysis and micro-CT analysis have strengths and limitations when evaluating bone regeneration. However, when combined, the findings can not only be validated by comparing the two methods but additional insight into the regenerative process can be gained. The purpose of the study was to gain FDA clearance for SteinerBio dental bone grafts for use in the skeleton.

A nonclinical GLP study was performed by creating critical size defects in the tibia of New Zealand White rabbits. Prior to sectioning for histological analysis, the samples were sent to the University of Michigan for micro-CT analysis. After micro-CT analysis, the samples were sent to HistoTox Labs for histological analysis.

Negative Control:

  • No graft


Positive Control:

  • Allograft


Test Materials:

  • Sinus Graft (marketed as Skeletal Graft Flow)
  • Socket Graft Putty (marketed as Skeletal Graft Putty)
  • Socket Graft Plus (marketed as Skeletal Graft Plus)

Osteotomies prepared

Grafted with Socket Graft Putty and Socket Graft Plus

Distinct Identity of Test and Control Articles and Doses of Articles

Each tibia is being divided in half between the osteotomies so that each sample includes only one bone defect.

The histological findings were produced by HistoTox Labs, an independent GLP laboratory specializing in research analysis.

TABLE 1. Mineralization Scoring Methods
HistoTox Mineralization Findings

Mineralization: Six to Eight Weeks Timepoint

Group mean score for the amount of bone at implant sites was highest in sites implanted with Skeletal Graft Flow and lowest in sites implanted with Allograft. Group mean scores for amount of bone at implant sites containing other implant materials (Skeletal Graft Plus and Skeletal Graft Putty) were similar to each other, but slightly lower than the group mean score for Skeletal Graft Flow (see below). All SteinerBio grafts produced more bone than the no graft negative control. A notable observation at the six to eight weeks timepoint was high scores for inflammation in sites containing Allograft:

As stated, the micro-CT analysis was performed at Michigan University Core research laboratory.

Definitions:
  • Bone volume (BV): measures the percentage of the tissue sample that is composed of mineralized tissue.
  • Bone Mineral Density (BMD): This parameter relates to the amount of bone within a mixed bone-soft tissue region but does not give information about the material density of the bone itself.
  • Tissue Mineral Density (TMD): the density measurement restricted to within the volume of calcified bone tissue excluding the surrounding soft tissue. This gives us information about the material density of the bone itself and ignores surrounding soft tissue.

Michigan Dental School core research laboratory raw data:

Averages of the raw data for Bone Volume, Bone Mineral Density, and Tissue Mineral Density:

Typical micro-CT images:

Performance Discussion
Histological evaluation can distinguish between newly formed mineralized tissue and residual graft material. However, the micro-CT scan does not distinguish between vital and nonvital mineralized tissue and only computes the total amount of mineralized tissue present in the tissue sample. Irrespective of this limitation, valuable information can be derived from the micro-CT scan data.

The histological evaluation performed in the nonclinical GLP study on rabbit tibia found that the commonly used positive control freeze-dried bone allograft produced the least amount of newly formed mineralized tissue. The histological findings showed that the allograft produced less mineralization than the no graft negative control documenting a negative effect on new bone formation. All SteinerBio graft materials outperformed both the allograft and the no graft negative control, documenting that SteinerBio bone grafts stimulate osteogenesis. In the micro-CT scan data, the allograft sites were found to contain more mineralized tissue by volume when compared to the negative control (no graft). However, the reason for this is that the micro-CT scan data counts both the new bone formation and the residual mineralized allograft particles.

When looking at the micro-CT scan data for bone volume (BV), the amount of mineralized tissue found in the control site is approximately half that of what was found in the allograft site. However, from the histological findings we know that the there was more new bone formation found in the control sites than in the allograft sites, indicating that more than half of the allograft sites are composed of allograft granules. From the histology findings we know there was no residual graft material found in the Socket Graft Putty and Sinus Graft samples, therefore, these SteinerBio grafts produced approximately twice the amount of mineralized tissue over the control and allograft. The bone mineral density (BMD) averages indicate that the amount of mineralized tissue produced by SteinerBio grafts were approximately 3 times the mineralized tissue produced by the control. Knowing that over 50% of the allograft sites were composed of residual allograft granules, the BMD data indicates that SteinerBio bone grafts produced greater than 3 times the amount of mineralized tissue over the allograft sites.

While the BMD data shows greater mineralized tissue volume produced by SteinerBio grafts, the tissue mineral density averages show that the mineralized tissue produced by SteinerBio grafts were superior not only in bone volume to the control and the allograft, but the mineralized tissue produced by SteinerBio grafts where superior in mineral density. This finding indicates that SteinerBio grafts not only produce a greater amount of mineralized tissue, but the mineralized tissue produced by SteinerBio grafts also have superior mineral strength.

Clinical Relevance

The histological and micro-CT scan findings in this study clarify a number of issues we have cited regrading socket grafting. The human body does not regenerate itself when wounded. These findings clearly show that not grafting produces far less mineralized tissue and that the mineralized tissue that is produced is inferior in quality to regenerated bone. The point is that not only do you lose significant ridge dimension when you do not graft, but the bone you are placing your implant in is inferior in both the amount of mineralized tissue present and the strength of the mineralized tissue.

Whenever you drill into poorly mineralized bone, it is always in nongrafted sites and this has been shown to compromise stability and longevity of dental implants. From this study, we know that allograft sites contain less vital mineralized bone than nongrafted sites, but more mineralized tissue owing to the presence of residual allograft particles. While the combination of newly formed bone and retained allograft particles give the radiographic and clinical impression of solid bone, in fact there is a minimal amount of actual vital bone to support the dental implant in allograft sites. From the findings of this study, allografts produce less bone than no graft, but the allograft also produced an intense inflammatory response that is responsible for the inferior bone volume and inferior bone quality. It is the combination of these negative factors that lead to a higher rate of implant complications in augmented sites containing cadaver graft materials. To our knowledge, there have never been any clinical study that has shown cadaver bone graft outperforming even the oldest synthetic bone grafts, and as science is always moving forward, cadaver bone grafts continue to be left further behind.

For those interested in a histological comparison of the various graft materials, please see the following. The histology below are representative samples of the animal study of the various experimental and control sites:
Skeletal Graft at 6 weeks. Woven bone is being remodeled by basic multicellular units into lamellar bone. 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 Skeletal Graft. The regenerative process is complete and the osteotomy is filled with mature cortical bone.

Skeletal 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:
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-power 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 osteoclasts.

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 8 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. 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 Skeletal Graft Plus. 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 Skeletal Graft Plus, 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.

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