Currently, human tissue regeneration still remains one of the highest challenges in both cell biology and translational research. The focus of the research involving human tissue regeneration has expanded to include reconstructive techniques requiring tissue sources, such as autologous/autogenic, homologous/xenogeneic donor sites, or heterologous processing systems, to restore small and large structural defects resulting from acquired and/or congenital pathological conditions affecting a variety of anatomical districts.
Research efforts have been directed at finding safe solutions to improve the risk/benefit profile when considering the use of tissue from autologous donor sites, and in particular, to reduce the functional compromise resulting from the use of autologous tissue donor sites.1,2 Clinical evidence continues to show that both tissue grafting, as well as free vascular flaps reconstructive procedures, are associated with a considerable risk of failure.3 In the past few years, efforts in biological tissue research and development have been directed at finding alternative tissue resources for bone reconstruction and regeneration in oral and maxillofacial surgery, as well as craniofacial surgery, and in particular, for the treatment of bony defects of the maxilla and the mandible.4
Despite the significant improvement in the surgical techniques for the treatment and reconstruction of maxillary pathology, which has indeed been associated with improved clinical outcomes, controversy continues to exist with respect to which bone source offers better and more predictable long-term viability for the reconstructive procedures required to correct maxillary pathology. Metabolic mechanisms, such as osteogenesis, osteoinduction, and osteoconduction, are not equally and optimally expressed in either autologous/autogenic or homologous/xenogeneic bone, as well as in heterologous biomaterials, thus the transplanted bone graft, independent of its origin, lacks optimal osteointegration.5
A recent review of tissue biotechnology engineering studies has provided evidence of the utility of tissue implantation to enhance the success rate of regenerative procedures.6 Cellular proliferation guided by a scaffold substrate and biological cellular mediators are the core elements in this process, thus the milestone concepts for cell tissue regeneration to possibly improve bone graft viability (Fig. 1). A recent study has identified a primitive human cell population called monocyte-derived multipotential cells (MOMCs) presenting with a fibroblast-like morphology and a unique phenotype featuring CD14, CD45, and CD34 receptors (Fig. 2).7 This novel cell type has mixed morphologic and phenotypic features similar to monocytes, endothelial cells, and mesenchymal cells. MOMCs are derived from circulating CD14 monocytes, and their differentiation requires binding to fibronectin and exposure to one or more soluble factors derived from peripheral blood CD14 cells. MOMCs comprise progenitor cells that can differentiate into a variety of nonphagocytic cells, including osteoblasts, fibroblasts, chondrocytes, adipocytes, and cardiac muscles cells, as well as neurons and endothelial cells, suggesting that circulating monocytes are more multipotential than previously believed (Figs. 3 and 4).6 Two clinical studies have documented the beneficial role of marrow-derived monocytes in enhancing healing of ischemic tissue loss secondary to peripheral arterial disease by improving neo-angiogenesis and overall perfusion of ischemic limbs.8,9 The key role of mesenchymal cell lineage, such as monocytes, both bone marrow or peripheral blood–derived, in neo-angiogenesis, enchondral, and membranous ossification,10 as well as in osseointegration and bone regeneration,11 has been expanded from its application to ischemic tissue loss secondary to vasculopathy to maxillary bone regeneration with preliminary encouraging results.12
The main objective of this study is to share our experience of an integrated treatment modality that includes the implantation of autologous monocytes and endothelial precursor cells in the bone graft itself at the time of reconstructive procedures of the maxilla and mandible from the standpoint of improved integration of the bone graft, as well as its long-term viability.
Materials and Methods
Following evidence that the use of peripheral blood monocytes and endothelial precursor cells was associated with neo-angiogenesis during the treatment of both ischemic and diabetic ischemic tissue loss (Institutional experience of the Department of Vascular Surgery), we decided to assess whether the use of autologous peripheral blood monocytes and endothelial precursor cells could be beneficial for standardized bone grafting surgical techniques from the standpoint of bone graft incorporation and long-term viability.
In 2015, we performed 19 bone grafting regenerative procedures in 11 patients affected by maxillary and mandible bony atrophy and defects of different severity secondary to atrophy or trauma. The procedures are summarized in Table 1, with a sample illustrated in Figure 5. We performed the following procedures: 8 sinus lift procedures, 6 performed with cellular therapy, and in 2 patients, we performed a “split mouth” comparative model; 5 posterior mandible on-lay bone grafting; 4 posterior mandible in-lay bone grafting; and 3 anterior maxilla on-lay bone grafting from the iliac crest.
Patients were thoroughly educated about the intended procedure, and informed consent was obtained. The perioperative care protocol is shown in Table 2.Tables 3 and 4, used as checklists in the operating room (OR), describe the individual steps for the procedures. An external observer monitored surgeons' workflow and the adherence to each surgical step. Surgical regenerative procedures were performed in all cases with standardized bone grafting techniques by qualified and experienced maxillofacial surgeons.
Description of the sinus lift technique
For sinus lift, bovine xenogeneic particle graft (Copioss by Zimmer) was used; only patients with no intraoperative complication (such as Schneider membrane perforation) were included in this study.
Description of the maxillary bone-block graft technique
In the case of maxillary regenerative procedure requiring bone-block graft, the dimensions and shape of residual bone and the dimension of the bony defect required an on-lay bone-block graft technique.13
Introduction to mandible regenerative techniques
For bone regenerative procedures of the posterior-lateral mandible segments, the extent of the atrophic defect could only be restored with a sufficiently large bone graft, namely a large cortical-medullary iliac crest bone-block graft (the proportion between defect and bone graft volume dictated the use of the iliac crest as donor site).14
Mandible regenerative techniques were selected based on the vertical residual volume detected by CT DENTASCAN between the alveolar canal and the edentulous alveolar ridge of the posterior-lateral mandible segments. Based on available evidence,15 we opted for an on-lay grafting technique if the vertical measurement from the alveolar canal to the edentulous alveolar ridge was less than 6 mm.
Description of the in-lay mandible technique
A longitudinal incision is performed on the vestibular aspect of the mucosal layer, followed by a longitudinal corticotomy executed at an average distance of 2 mm from the top of the alveolar canal; separation of the crestal part from the residual basal bone is obtained and lifted upward assuring attachment of the segment to the periosteal surface, thus the space obtained by vertical displacement becomes the recipient site of the cortical-medullary iliac crest bone-block graft.
Description of the on-lay mandible technique
On-lay technique is performed by directly positioning and stabilizing the cortical bone-block graft on the cortical surface of the residual edentulous ridge.
Cell Harvesting and Their Application
An approved medical device,16 functioning as an automated, filtration-based point-of-care blood cell separator, designed to obtain a high concentration of peripheral blood monocytes and endothelial precursor cells by gravity filtration, has been used in the OR in the past for other tissue regenerative procedures without any bone marrow cell mobilization protocol or granulocyte colony-stimulating factor (G-CSF) use.9,17,18 The same device has been used to enhance the treatment of ischemic and diabetic ulcers.16,19
The steps required to retrieve the monocytes and endothelial precursor cells are summarized in Figure 6. In brief, 60 mL of peripheral blood sample is drawn with a heparinized syringe and collected into the first-collect bag unit (1) and subsequently filtered through one of the system components (2). At the end of the filtration process, monocytes and endothelial precursor cells remain entrapped in the filter component (2), and the remaining blood cellular elements are collected into the lower disposal bag (3). At this point, the filter is flushed with 10 mL of sterile saline through the “port C” of the system, thus obtaining 10 mL of cell concentrate (a mean of 2.46 ×106 cells) in the cell collection bag (4): 2 mL is used for the specific procedure. To assess the level of cellular enrichment achieved, we evaluated the ratio of white blood cell count from the remaining 8 mL and compared it with the patient's white blood cell count.
All procedures were performed by coating the bone graft, (either autologous/autogenic, homologous/xenogeneic, or heterologous) after having placed it at the site of the defect/atrophic area, with a platelet-rich plasma (PRP)-Gel matrix obtained by centrifugation of the initial blood sample, which was then used as a substrate/scaffold to implant and stabilize the MOMCs and endothelial cell precursors at the grafting site. This was achieved by seeding the PRP gel coating, by simply inoculating it with randomly distributed drops of the cell concentrate using a 1-mL small needle syringe to minimize cell dispersion (Fig. 7).
The following definitions were used to assess the effectiveness of our methodology with respect to sinus lift procedures and both on-lay and in-lay bone-block grafts.
Sinus lift cases
- Graft reabsorption was defined as more than 10%-dimensional reduction of the original graft volume at 1-year follow-up.
- Failure was defined as a graft loss resulting in inability of fixture insertion at the second stage of surgery, 3 to 12 months after sinus grafting.
- Survival of fixtures was defined as fixture remaining in situ during the entire observation period.20
- Graft reabsorption was defined as more than 30%-height reduction in the grafted area at 1-year follow-up.
- Failure was defined as a graft loss resulting in inability of fixture insertion at the second stage of surgery, 3 to 12 months after grafting.
- Survival of fixture implant was defined as a fixture remaining in situ during the entire observation period.21
- Bone density was evaluated based on Hounsfield CT units.22
In the first 3 weeks after surgery, there were no complications (surgical sites did not present dehiscence; there were optimal mucosal flap tropism and approximation; and no evidence of local infection).
The patients underwent CT DENTASCAN 1 day after surgery, before fixture placement (8 months after regenerative procedures) and at 12 months after fixture placement.
Of note, the signal detected in the bone graft sites showed significant differences when the patient's treated with particle xenogeneic bone chips implanted with monocytes were compared with patients treated with particle xenogeneic bone chips but without monocytes. Additional differences were noted when patients with similar defects treated with xenogeneic bone chips with monocytes were compared with those treated with a segment of the crest cortical/medullary autologous bone graft with monocytes.
All procedures performed with xenogeneic particle bone graft and cellular therapy resulted in poor and disorganized radiological density signal suggesting poor osteogenesis and an inflammatory reabsorption of the graft (an average of 80% of the original graft volume). In comparison, the 2 cases treated with a “Split Mouth” left/right sinus lift procedures with xenogeneic particle bone graft but without cellular implantation showed a regular and uniform radiological signal suggestive of healing with good osteointegration.
Four patients with a mean defect volume of 2.2 cm3 on each side underwent bilateral sinus lift with xenogeneic bone grafts with cellular therapy. Two of the 4 patients were treated with cellular therapy bilaterally, and the remaining 2 had one of the sides treated without cellular therapy. This allowed one side to be used as the control side to assess the impact of cellular therapy on bone reabsorption.
The 2 patients treated with bilateral sinus lift with cellular therapy suffered a 2/3-dimensional reabsorption at 18 months, whereas the remaining 2 patients with differential treatment, namely one side treated with cellular therapy and the other without it, had a 2/3 reabsorption on the side treated with cellular therapy and a low reabsorption on the side treated without it (Fig. 8).
Eight patients were treated with either on-lay or in-lay bone grafts from the iliac crest or the mandible with cellular therapy to correct defects ranging in size from 1.5 × 1.5 × 1 cm to 4.5 × 2.5 × 1.5 cm of the maxilla or the mandible.
All patients treated with bone-block graft implemented with monocytes showed no appreciable reabsorption of the bone graft block at 18 months (Fig. 9).
One patient, a 39-year-old woman, treated with both a left maxillary on-lay graft from the mandibular ramus and a right maxillary sinus lift graft with implantation of mononuclear cells showed no reabsorption on the left side (perfect healing of the block graft) but, again, an 80% reabsorption of the sinus lift side on the right consistent with the failure of the sinus lift.
In randomly selected cases, we evaluated digital bone density of mandible-grafted sites by means of an implant planning software (SimPlant by Materialise), confirming the cortical bone formation of the autologous bone graft (Fig. 10). At 1-year follow-up, there was no fixture survival in sinus lift sites implemented with cellular implantation, whereas fixture survival was 100% in the cases of atrophic maxillary bones grafted with cortical-medullary iliac crest bone block, performed with either in-lay and on-lay techniques. These results remained unchanged at 18-month follow-up (Table 1).
For preparations, the bone cores were fixed in 10% neutral-buffered formalin and sent to the histo-biology research laboratory at Catholic University of Rome. On receipt, there the specimens were immediately dehydrated with a graded series of alcohol for 7 days. After dehydration, each specimen was now fixed and embedded in paraffin at room temperature never exceeding 25°C. The specimens were then cut to a thickness of 18 μm and then stained with hematoxylin and eosin. Finally, histologic analysis was obtained by means of dry-field and polarized microscopic evaluation.
Fan beam computed tomographay/cone beam computed tomography (FBCT/CBCT) data and our clinical observation were confirmed by histological examination of bone specimen harvested from the grafting sites at the time of implant placement from both sinus lift and block graft. Figure 11 representing bone sample histology from both on-lay and in-lay iliac crest grafts shows an organized pattern in the laminar bone, contributing to the initial formation of primary osteons, with neo-angiogenesis and maturation of Havers and Volkmann canals, similar to the native mandible one. The impact of the implantation of monocytes on the sinus lift graft can be seen in the histological evaluation. Sinus lift graft without implantation of monocytes shows a histological pattern that although not perfectly organized is similar to the native bone. By contrast, the sinus lift graft performed with the implantation of monocytes displays a very disorganized pattern that suggests the presence of a mixed inflammatory fibrosis and osteoid matrix. The implantation of monocytes in sinus lift using particle xenogeneic bone chips slows healing when compared with treatment without cellular implantation. In addition, its use is associated with a disorganized healing pattern, featuring an intense and prolonged inflammatory phase, thus preventing osteoid matrix deposition. Such condition may interfere with bone formation and may compromise optimal implant osteointegration.
Despite the significant improvement in the surgical techniques for the treatment and reconstruction of maxillary pathology, which has indeed been associated with improved clinical outcomes, controversy continues to exist with respect to which bone source offers better and more predictable long-term viability for the reconstructive procedures required to correct mandibular and maxillary pathology. Both maxillary and mandibular defect sites can be treated with standardized bone grafting techniques, ranging from the on-lay bone grafting technique with cortical-medullary autologous block (iliac crest) and in-lay bone grafting sandwich technique also with cortical-medullary autologous block (iliac crest) to the basic sinus lift with xenogeneic particulate cortical and medullary bone, considered by some authors as an in-lay graft.23
The selection of autologous bone graft donor sites is predicated on many factors, including the ratio of the bone defect to the volume of the bone graft required which is in turn related to the survivability of the bone graft and the final long-term results from the standpoint of the implantation of fixtures.24 In our experience and that reported by other authors, different bone grafting techniques have shown different results for bone graft osteointegration and osteoconduction in terms of bone quality and volume obtained, and ultimately long-term survival. Clinical evidence of longer bone graft survival and better cortical bone formation process has been reported when in-lay and on-lay iliac crest cortical-medullary bone graft techniques have been compared.21
Among all commonly used autogenous bone grafts, the iliac crest, based on our experience and that reported in the literature, shows a less optimal osteoconductive metabolic response and long-term survival from the standpoint of bone volume and quality of cortical bone density when compared with bone grafts harvested from mandible donor sites or cranial calvarial bone.25,26 The iliac crest still remains one of the most common large-volume autogenous bone graft donor sites; however, because of its intrinsic histology, namely poor cortical cancellous fraction, it does not have the ideal long-term survival when compared with bone grafts raised from other donor sites, such as the symphysis or the ramus of the mandible. The latter site in fact, although does not provide large bone volume, is superior from the standpoint of osteogenesis and osteoconduction because of the well-expressed cortical cancellous fraction according to our experience and that of other authors.27
Based on many of the limitations associated with the conventional bone grafting procedures, mainly from the standpoint of long-term outcome, and based on the successful use of monocytes and endothelial precursor cells in the treatment of ischemic tissue loss, we decided to compare the outcome of the most predictable regenerative procedures (sinus lift) with the less predictable ones (on-lay and in-lay autologous bone grafts from the iliac crest) when implemented with and without MOMCs. Thus, we applied this new regenerative concept for the treatment of maxillaries' bone defects and atrophies with autogenous cortical-medullary iliac crest block graft and used the common sinus lift procedures implemented in the same manner as “control cases.”
Our results show that the addition of monocytes and endothelial precursor cells to the grafting techniques using iliac crest autologous cortical-medullary bone block facilitates bone graft osteointegration by promoting in-site bone graft revascularization, thus leading to improved long-term results. The postulated mechanism of the beneficial effect of peripheral monocytes and endothelial precursor cells in the setting of an already structured bone (primary and secondary osteons), such as the cortical medullary block graft on osteointegration, involves enhanced angiogenesis through the pre-existing collateral vessel network represented by Havers and Volkmann canals, starting their reorganization in a more functional and competent structure.28
Our point-of-care (POC) filtration system, which allows cellular harvesting during the surgical procedure, provided a 394% enrichment consistent with what has been previously reported29 (Fig. 12). This level of enrichment resulted in a 4 times higher level than that reported by other authors.12 Of note, our filtration, as well other filtration methods used to harvest cellular material from peripheral blood are not selective for monocytes filtration. Cell-specific selection devices require a much more complex system not compatible with POC use in an OR setting.29
Our results of sinus lift procedures performed with the implantation of monocytes differ from those reported by Soltan et al12 and others30,31 with respect to the detrimental effect of monocytes seen in our patients.
Several observations indicate the presence of 2 subsets of monocytes: resident and circulating ones. The resident tissue macrophage populations are mostly derived from the yolk sac during embryogenesis; fetal liver and hematopoietic stem cells contribute to macrophage production only at a later time.32,33
These tissue macrophages play critical roles during development and also provide important trophic signals that support neighboring parenchymal tissues and are critically involved in normal tissue homeostasis.34
The role of resident tissue macrophages versus recruited monocytes has become an important area of research, as there is accumulating evidence that different monocyte and macrophage populations play distinct and nonredundant roles in tissue repair, fibrosis, and regeneration35
The mechanisms that instruct macrophages to adopt proinflammatory, prowound healing, profibrotic, anti-inflammatory, antifibrotic, proresolving, and tissue regenerating properties in various organ systems have also been the subject of intensive research.36,37
It is unclear whether an individual macrophage (local or recruited) is capable of adopting all of these attributes at different times or whether there are truly distinct functional subsets.
An experimental study on mice through genetic fate mapping assessment demonstrated that most macrophages in the heart of an adult-testing subject are derived from the yolk sac and fetal progenitors; differently, the dominant macrophages driving the early inflammatory response in cardiac tissues after injury are C-C chemokine receptor type 2 presenting ones (CCR2+ monocyte or circulating ones). In this study, the suppression of circulating monocytes recruitment through genetic manipulation led to a large preservation of tissue macrophages, reduced inflammation, and accelerated repair.38
In another study, these 2 cellular subsets seem to represent different evolutionary stages in which monocytes proceed and evolve in tissue macrophages; the latter cell population would remain in the healed tissue and participate in its homeostasis; pathway and factors regulating this evolution in vivo are still unknown.39
Difference between our results and the ones by other authors may be in part explained due to the higher concentration of monocytes in our preparation that may have caused a more intense inflammatory response responsible for the reabsorption of the particle graft. An additional reason may include the difference in vitality at the time of inoculation and the survival rate of monocytes between our harvesting method and that reported by Soltan. However, it is unknown whether the results depend on monocyte concentration, their vitality and survival rate, or on the presence of other cell populations and their specific concentration that can be present when harvesting is performed without a specific self-selected device. Another reason for the difference in the results of our study as opposed to others involves the effect of different source of graft material on sinus lift procedures.40
We believe that, based on the current absence of evidence of metabolic superiority of bone marrow–derived versus mobilized peripheral blood cells, and in consideration of the potential complication arising from bone marrow–derived cell harvesting procedures, such as local pain, hematomas, and anemia,19 it is advisable to use peripheral blood–derived cells without G-CSF-induced mobilization. Peripheral blood mononuclear cells are an easily accessible cell source for cell therapy noninferior to bone marrow mononuclear cells. Notably, patients treated with peripheral blood–derived cells have a higher mononuclear cell fraction as opposed to that from bone marrow.20
The filtered cells obtained from the device used in our procedures have a comparable and significant migratory ability, indicating that the device filtration does not impair the ability of cells to migrate on chemoattractant stimuli or their viability. In addition, the supernatant obtained from the filtered cells demonstrates levels of 10 tested angiogenetic cytokines significantly higher than that for Ficoll separation29 (Fig. 13). Our filtering system has the following advantages: (1) it is cost effective; (2) it is a point-of-care system that can be used on site in the OR; and (3) it provides a high concentration of peripheral blood monocytes.
Although G-CSF has proved effective in causing a large mobilization in the bloodstream of bone marrow cells, concerns about rare but possible serious adverse events have prompted researchers to test cell harvesting from peripheral blood in the absence of stimulation.16,40 Peripheral blood monocytes harvested without G-CSF-induced mobilization have been shown to improve angiogenesis in 42 patients with chronic limb ischemia.16
At this time, it remains unclear which is the best volume and cell concentration needed to enhance healing of tissue and bone graft material. Experimental studies have demonstrated that a volume of 120 mL of peripheral blood and a cell count of 1.06 × 108 mononuclear cells may be sufficient to regenerate soft tissue in ischemic wounds.41 We opted in our study to proceed with blood volume of 60 mL, yielding a cell concentration volume of 10 mL and a cell count of 2.46 × 106, to study its effect on bone integration and long-term reabsorption.
Based on the results of our observational study, we conclude that the implantation of monocytes and endothelial precursor cells may be beneficial in enhancing healing and long-term survival of the autologous bone graft, but it may be detrimental when used in combination with xenogeneic particle bone graft. We believe that the results of our study warrant further investigation in a multicenter prospective randomized control trial properly powered to answer the question of whether the use of peripheral blood mononuclear cells should become an integral part of an integrated approach to the treatment of maxillofacial conditions requiring surgical reconstructions with bone grafts.
Our study has many limitations which obviously prevent generalization of our conclusions. They include the following:
- A small nonhomogeneous sample size
- A difference in the type and size of the defects treated
- The inability to perform any statistical analysis in view of the limited sample size
- Lack of histochemistry and microCT assays
The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.
There is no assigned IRB number, but a research registry number (#researchregistry2689#).
Roles/Contributions by Authors
Vito Del Deo: surgeon. Antonio Fico: study on therapeutic angiogenesis biological principles and application; external observer of the surgeons' workflow; and responsible for all diagrams in the manuscript. There is no conflict of interest for all of them nor are any of them commercial. Corrado Marini: study on therapeutic angiogenesis clinical application. Salvatore Senese: cell concentrate harvesting. Giulio Gasparini: surgeon.
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