A Histomorphogenic Analysis of Bone Grafts Augmented With Adult Stem Cells

Abstract

Purpose: 

To evaluate the influence of bone marrow aspirate added to xenograft or alloplast graft matrix scaffold to produce bone.

Materials: 

A maximum of 4 cc bone marrow was aspirated from the anterior iliac crest of 5 patients to saturate the matrix scaffold prior to bone graft. Seven graft sites evaluated included sinus lift augmentation, particulate onlay graft of the maxilla via a tunneling procedure, and particulate onlay graft of the maxilla stabilized with titanium mesh. The xenograft scaffold was either PepGen Putty (DENTSPLY Friadent CeraMed, Lakewood, CO) or C-Graft resorbable algae material (Clinician’s Preference, Golden, CO). The alloplast scaffold was β-tricalcium phosphate (either Curasan AG, Kleinostheim, Germany, or Vitoss; Malvern, PA).

Results: 

Graft sites healed for 4–7 months. Core specimens of graft sites were taken with trephine drills, and submitted for standard histologic and histomorphogenic analysis. The percentage of graft material converted into bone, percentage of vital graft matrix, percentage of unresorbed matrix, and percentage of remaining interstitial tissue were measured. After a 4-month healing of sinus-lift augmentation with C-Graft, the biopsy showed 31% bone that was 100% vital. Unresorbed graft material was 26%, and remaining interstitial material constituted 43%. Using pure phase β-tricalcium phosphate, a 4-month core biopsy showed 40% bone that was 100% vital. Residual graft was 3% and interstitial material 57%. A sinus grafted with PepGen P-15 (DENTSPLY Friadent CeraMed) was found to be 14% bone, with 100% of that bone vital. The non-bone within the core was 36%. After a 4 1/2-month healing of bilateral sinus grafts using a nonpure phase β-tricalcium phosphate, the percentage of the biopsy that was bone was 23% on the right side and 16% on the left side. Vital bone was 89% (right side) and 86% (left side). The core taken after 4 months of healing from the anterior maxilla particulate onlay graft with PepGen P-15 showed 32% bone, with 100% found to be vital. Non-bone within the core was 15%, and 53% was interstitial material. After 7 months of healing, a biopsy core from the maxillary ridge augmented with C-Graft was 45% newly formed bone, with 100% of the bone vital. There was no residual graft material present.

Discussion: 

Bone regeneration by cell-based strategies depends upon an understanding of the biology and potential of adult stem cells as a method of regenerating bone.

Conclusions: 

Bone marrow aspirate containing adult stem cells when mixed with bioengineered graft materials provide a scaffold to support the proliferation, differentiation, and maturation of the stem cells, as well as facilitating angiogenesis. This article presents histological evidence that stem cells aspirated from bone marrow and transplanted onto biocompatible scaffolds can successfully regenerate bone. This new standard for bone grafting may emerge as an alternative to autogenous bone grafts.

Several conditions must be present for any bone graft to succeed. The graft site must contain osteogenic cells, in particular osteoblasts, the only cells that directly form bone. Without osteoblasts or the precursor cells from which they form, the process of osteoinduction cannot occur. The graft site must also contain a resorbable scaffold to enable new bone to form by osteoconduction. The grafted scaffold later is resorbed via a cellular-mediated reaction. Soluble regulators, acquired from the blood, adjacent tissue, or from stem cells found in bone marrow, are also essential. The need for their presence at the site is one reason why the establishment of an adequate blood supply is crucial. Achievement of a tension-free closure of the surgical site helps to ensure that the blood supply will be adequate and protects the graft from the oral environment.

An understanding of the mechanisms of bone growth (osteoconduction, osteoinduction, and osteogenesis) can facilitate the selection of the optimal grafting material for any given situation.¹ Available graft materials differ in the mechanisms through which they influence host-bone formation. Freshly harvested autogenous bone brings a rich mixture of osteogenic cells, osteoconductive scaffold material, and soluble regulators to the graft site.² For this reason, autografts have long been considered to be the gold standard. However, all autografts require a second surgical site, with the attendant risks of morbidity.

To avoid the need to harvest autogenous bone, a variety of biocompatible alternatives have been developed, including allogenic, xenogenic, and alloplastic materials. These materials all provide the graft site with a biocompatible scaffold. None, however, contain live cells.³ Their success has always depended upon the availability of osteogenic cells and other biologic-regulating agents from the peripheral host bone and blood.^4–6

Adult Stem Cells

An increased understanding of the nature and role of adult stem cells has introduced the possibility of regenerating bone from allogenic, xenogenic, and alloplastic materials to an extent that approaches or even exceeds the results obtainable with autografts.^7–9 Stem cells by definition are capable of both self-renewal and differentiation into a variety of mature cell types. Stem cells derived from bone marrow include hematopoietic and mesenchymal stem cells.^10–13 Transplantation of marrow cells, therefore, gives rise to hematopoietic and osteogenic lineage cells. Hematopoietic stem cells augment the limited number of available stem cells to support angiogenesis and vasculogenesis. Osteoblastic precursors can differentiate into the mature osteoblasts that are needed to promote osteogenesis.¹⁴ Marrow cells promote osteogenesis. Evidence that they contribute to the success of grafts in osseous defects is compelling.^15–17

Recognizing the potential inherent in combining bone-marrow stem cells with existing allogenic, xenogenic, and alloplastic scaffold materials, the authors have developed a technique for aspirating bone marrow from several sites.¹⁸ Simple and painless, this technique eliminates the need for a second surgical site. Morbidity is minimal.

The focus of this report is an in vivo study of adult stem cells derived utilizing this technique, then mixed with either an allograft, xenograft, or alloplast scaffold. The study objective was to evaluate histologically the extent of new bone growth at such sites.

Materials and Methods

Seven graft sites in 5 patients were evaluated. The xenograft scaffold was either PepGen Putty (DENTSPLY Friadent CeraMed, Lakewood, CO) or C-Graft resorbable algae material (Clinician’s Preference, Golden, CO). The alloplast scaffold was β-tricalcium phosphate (either Curasan AG, Kleinostheim, Germany or Vitoss; Malvern, PA) Three of the patients were grafted using sinus-lift subantral augmentation.^19,20 The sinus-lift surgery protocol was as follows.

Each patient was prepared and draped, then 2% Xylocaine (Cook-Waite, Abbot Laboratories, Chicago, IL) with 1:200,000 epinephrine was locally injected. After adequate local anesthesia was achieved, a crestal incision and vertical relief incision were made. A mucoperiosteal flap was reflected along the crestal alveolar bone, superiorly to the height of the vestibule. The flap exposed the region of the canine fossa, malar buttress, and region of the pterygomaxillary fissure and the tuberosity. The procedure continued with the development of a quadrilateral osteotomy. After lifting the sinus membrane, the graft material (either peptide-enhanced anorganic bovine particles, resorbable algae-derived material, or β-tricalcium phosphate) was mixed with bone-marrow aspirate and inserted. The flap was then repositioned and sutured.

The fourth patient received an onlay particulate graft. For this procedure, a mucoperiosteal flap was reflected exposing the labial cortical bone. Decortication of the labial plate was accomplished. A titanium mesh was secured with screws on the palate. A guided bone regenerative membrane was placed under the mesh, and under the membrane, a loose compaction of bone-marrow aspirate mixed with PepGen P-15 (DENTSPLY Friadent CeraMed) anorganic bovine putty was deposited. The titanium mesh was then secured with screws on the labial, and the periosteum was relieved to obtain primary closure without tension at the incision. The site was sutured with 3-0 Vicryl material (Ethicon, Inc., Johnson & Johnson, Somerville, NJ).

The fifth patient’s left maxillary ridge in the canine-bicuspid area was grafted using a tunneling technique. A vertical mucoperiosteal incision was made, and a subperiosteal tunnel was developed. A composite graft consisting of bone-marrow aspirate mixed with loosely compacted C-Graft resorbable material was injected through the tunnel.

In each case, the bone marrow was extracted from the patient using the technique described in detail by Smiler and Soltan.¹⁸ This technique can be performed as an outpatient procedure with the patient under oral sedation and local anesthesia, intravenous sedation, or general anesthesia. The bone marrow can be extracted from the sternum, posterior ilium, or anterior iliac crest. A bone-marrow aspiration needle is held with the index finger near the tip to control the depth of penetration. The needle is then advanced with steady pressure and a twisting motion through the cortical bone to approximately 1 cm inside the marrow cavity. (Decreased resistance indicates penetration into the marrow cavity.) The obturator/stylet is removed, and a 10-mL syringe is attached to the needle. Following aspiration of 2–4 ml of bone marrow, the needle is removed.

All 5 patients were allowed to heal for 4–7 months. After obtaining informed consent, specimens of the grafted areas were taken with trephine drills. These were then submitted to an oral pathologist for standard histologic and histomorphogenic evaluation.

The analysis measured the percentage of graft material that had been converted into bone, percentage that was unresorbed, and remaining interstitial tissue. In addition, all bone identified in the biopsies was assessed to ascertain the percentage of bone that appeared to be vital.

Results

Case 1

After sinus-lift surgery to create the graft-recipient site, bone-marrow aspirate was mixed with C-Graft resorbable material and deposited with loose compaction to reconstitute the buccal wall of the maxilla. After 4 months, a biopsy was taken with a trephine drill from the crestal bone to the superior aspect of the graft.

The percentage of the biopsy that was found to be bone was 31%. One hundred percent of that bone was vital. The percentage of unresorbed graft material within the core was 26%. Interstitial material constituted the remaining 43%. Within the cancellous bone pattern, all the graft particles were well incorporated.

Fig. 1 shows the overall view of the core and reveals the new bone formation bridging the graft particles. Remodeling of mature bone is evident. New bone formation with C-Graft and the pattern of the coral are seen at high power in Fig. 2.

Fig. 1.: Overall view of the core showing integration of graft and new bone formation bridging graft particles.Fig. 2. Shows new bone formation and C-Graft particle.

Case 2

Bilateral sinus-lift surgery was initiated, and the right sinus was loosely filled with pure phase β-tricalcium phosphate mixed with bone-marrow aspirate to reconstitute the lateral wall. The left sinus recipient site was grafted with PepGen P-15 anorganic bovine particulate. After 4 months, biopsies were taken with a trephine drill from the crestal bone to the superior aspect of the grafts.

The percentage of the right-sinus (β-tricalcium phosphate) core biopsy that was found to be bone was 40%. One hundred percent of that bone was vital. The percentage of non-bone within the core was 3%. The remaining 57% of the sample consisted of interstitial material. All the graft particles were well incorporated within the cancellous bone pattern. Fig. 3 shows good bone formation and distribution in the core biopsy. The bone appears to be very dense, with a good cancellous pattern incorporating well-formed trabeculae, connective tissue, and graft material particles. Tricalcium phosphate particles are embedded in the newly formed bone with evidence of osteoid and osteoblasts (Fig. 4).

Fig. 3.: Core biopsy shows good bone formation and distribution consisting of very dense trabeculae with cancellous pattern.Fig. 4. Tricalcium phosphate particles are embedded in newly formed bone with osteoid and osteoblasts.Fig. 5. There is significant tight compaction of the P-15 scaffold, leading to a decrease in space between particles and decrease in bone formation.Fig. 6. Very high-power shows PepGen P-15 particles embedded in bone, and new bone formation with osteoid and osteoblasts.

The percentage of the left-sinus (PepGen P-15) core biopsy that was found to be bone was 14%, with 100% of that bone found to be vital. The percentage of non-bone within the core was 36%. The tight compaction of the graft material that is evident in Fig. 5 suggests an explanation for the decreased bone formation. Tight compaction of the scaffold leads to a decrease in space between the particles and a decrease in bone formation between the particles. Fig. 6 is a high-power image showing new bone formation with osteoid and osteoblasts and PepGen P-15 interaction with bone. The PepGen P-15 particles are both embedded in and attached to the bone.

Case 3

Bilateral sinus-lift surgery was initiated, and both sinuses were grafted with β-tricalcium phosphate (Vitoss) mixed with bone-marrow aspirate. After 4 1/2 months, biopsies were taken with a trephine from the crestal bone to the superior aspect of both grafts. The percentage of the right-sinus biopsy that was found to be bone was 23%, with 89% of that bone found to be vital. The percentage of non-bone within the core was 13%. The percentage of the left-sinus biopsy found to be bone was 16%, with 86% of the bone found to be vital.

New bone formation and resorbing graft matrix within the right biopsy can be seen in Fig. 7. A high-power image (Fig. 8) shows viable cells within the lacunae of new bone formation in the right biopsy core.

Fig. 7.: Low-power image of core biopsy showing bone formation.Fig. 8. Very high-power shows viable cells within lacunae.

Case 4

This core was taken from the anterior maxilla particulate onlay graft with PepGen P-15 after 4 months of healing. The percentage of the biopsy that was found to be bone was 32%, with 100% of that found to be vital. The percentage of non-bone identified within the core was 15%. The remaining 53% consisted of interstitial material.

The core shows a sound cancellous bone pattern with new-bone formation independent of the particles of PepGen P-15 (Fig. 9). A few particles are incorporated into the new bone formation. A very high-power image (Fig. 10) shows new bone formation, which is attached to the PepGen P-15 particles.

Fig. 9.: A cancellous bone pattern with new bone formation independent of the PepGen P-15 particles is evident in this core.Fig. 10. Very high-power images show new bone formation attached to the PepGen P-15 particles.

Case 5

After 7 months of healing, a biopsy core was taken and found to consist of 45% newly formed bone. All of that bone was vital, and there was no residual graft material present.

Fig. 11 shows that the cancellous bone pattern is composed of very thick trabeculae. New bone formation with C-Graft and the pattern of the scaffold are seen at high power in Fig. 12.

Fig. 11.: Note the very thick trabeculae within the cancellous bone pattern.Fig. 12. High-power view of the new bone formation amid the C-Graft scaffold.

Discussion

Bone-graft healing involves the recruitment and proliferation of cells capable of restoring the graft site to its original form and function. Over the years, the success of bone-grafting procedures has only occasionally been evaluated using histomorphometric techniques.

Peleg et al²¹ reported on 156 sinus floor augmentations and 37 alveolar ridge grafts. Half the sinus augmentations were grafted with a 50% mix of Bio-Oss (Osteohealth, Shirley, NY) and surface-derived (cortical) autogenous bone. The other half was grafted with autogenous bone alone. The ridge augmentation patients were grafted with cortical autogenous bone. Biopsy cores of the grafts were taken between 4 and 8 months after healing. The core biopsies showed on average immature, newly formed bone and on average 27% to 36% vital bone.

Moy et al²² reported on a biopsy core taken from a single patient 25 months after sinus grafting with autogenous bone. They described the composition of the core as being 59.4% bone and 40.5% soft tissue.

Pejrone et al²³ reported on histomorphometric results of 6-month biopsy cores from 13 patients grafted with autogenous iliac bone blocks that showed a mean percentage of 54.1 mineralized tissue in the grafts.

Froum et al²⁴ reported on a core biopsy taken from 1 patient who had had a sinus augmentation with Puros mineralized cancellous bone allograft (Zimmer Dental Inc., Carlsbad, CA) mixed with autogenous bone. After 9 months of healing, the vital bone content was 25.2%, connective tissue and marrow constituted 58%, and 16.8% was residual graft material.

Hans-Dieter and Wenz²⁵ reported the analysis results from 21 patients whose sinus augmentation was with Bio-Oss natural bone mineral alone and 13 patients grafted with a combination of Bio-Oss and autogenous bone. Biopsies were taken from all patients 3–8 months after surgery. Histomorphometric analysis showed no statistically significant difference between the patients grafted with only Bio-Oss (overall amount of 29.52% new bone formation) and the Bio-Oss/autogenous bone group (overall amount 32.23% new bone formation).

In a comparative analysis of sinus augmentation with different biomaterials, Scarano et al²⁶ reported on the results of 94 patients. Each patient underwent biopsy after 6 months of healing. A total of 144 biopsies were retrieved. Those who had been grafted with autogenous bone showed an average of 40.1% newly formed bone, with residual graft particles averaging 18%. Those who received demineralized freeze-dried bone had an average of 29% newly formed bone, with 34% residual graft. Those who got PepGen P-15 had an average of 37% new bone, with 37% residual particles. Those grafted with Bio-Oss had an average of 39% new bone, with 31% residual particles.

Schopper et al²⁷ reported histomorphologic findings of 26 patients 6 months after bone augmentation with Algipore (same as C-Graft; DENTSPLY Friadent, Mannheim, Germany) in 1999. Histological examination revealed a physiologic framework of cancellous bone formed around Algipore biomaterial particles. The mean percentage of mineralized bone was 20.3%.

Szabo et al²⁸ in 2005 reported the results of using Cerasorb (β-tricalcium phosphate; Curasan AG) versus autogenous bone for bilateral sinus grafts of 20 patients. Analysis of biopsies taken at 6 months showed the mean percentage of bone for the Cerasorb to be 36.47%. The mean percentage for the autogenous bone was 38.34%. The difference between the 2 results was not statistically significant.

It is important to note that of the recent studies, only 2 studies^21,24 reported on what percentage of the total new bone was vital bone. That percentage ranged from 25.2% to 36%. All the other studies identified the percentage of mineralized bone or new bone formation but failed to distinguish the percentage of active living cells contained within the newly formed bone. In contrast, the current study identifies the percentage of viable bone. In 5 of the 7 sites biopsied, 100% of the bone was vital (containing living cells), while in the other 2 sites, the respective percentages were 89% and 86%.

In the 1 case where only 14% of the biopsy core contained mineralized bone, overcompaction of the grafting site was evident, an error that likely contributed to the limited success of the graft. In case No. 3, where Vitoss TCP was the graft material, the low percentages of new bone within the core (16% for the left sinus and 23% for the right sinus) might be explained by the physical properties of the graft scaffold. Vitoss granules have a porosity of about 90%. In contrast, Cerasorb granules have a porosity of 65%, making them more stable. The interconnecting pore distribution of Cerasorb is also narrower than that of the Vitoss particles. Furthermore, Cerasorb granules are pure-phase, while Vitoss granules have a foreign phase proportion of approximately 2%.

Another potential cause of variation in the results obtained when using stem cells may be the age of the patient. As patients age, their red marrow (rich in stem cells) decreases and is replaced by yellow marrow (poor in stem cells). Techniques for concentrating the stem cells for older patients are now being evaluated.

In 4 of the 7 cases included in the current study, the percentage of mineralized bone identified (between 31% and 45%) compares favorably with the mean percentage of mineralized bone found by Szabo et al²⁸ (38.34%) in sites grafted with autogenous bone. Furthermore, the use of stem cells aspirated from bone marrow eliminates the need for the creation of a second surgical site for harvesting autogenous bone.

Additional studies are needed to investigate how stem cells influence the development and repair of peripheral tissue. Markers for stem cells of different tissues and organs are currently under study.

Conclusions

Although preliminary in nature and limited in scope, this study demonstrates that the combination of stem cells (derived from bone marrow) with a scaffold material within a bone-grafting site can produce a significant quantity of viable bone. The therapeutic potential of bone regeneration by such cell-based strategies is highly promising.¹⁴ Stem cells derived from bone marrow are remarkably plastic and pluripotent, and they can roam throughout the body, taking up residence wherever they are needed to promote regeneration.^15,16

Disclosure

The authors are on the board of directors of the not-for-profit Platinum Foundation for Research and Education, which sponsored this research and manuscript. Curasan (Cerasorb) has made a financial contribution to the foundation. The authors claim not to have a financial interest in Curasan, or a financial interest in any company whose products are mentioned in this article.