Treating bone healing disorders represents a huge challenge for orthopedic and trauma surgeons and frequently produces unsatisfactory results. Critical size bone defects, in particular, which appear after tumor surgery or trauma do not heal spontaneously and require special therapy. There are also diseases which, despite surgical intervention and the application of all conventional therapies to promote bone regeneration, are accompanied by insufficient bone healing. These include aneurysmal bone cysts, enchondroma and congenital pseudarthrosis. In the broadest sense, bone defects also include avascular osteonecrosis which is defined by the death of osteoblasts. In addition to successful bone healing through the use of growth factors, increasingly positive results of osseous regeneration through stem cells have been published in recent years.1
This article describes the current state of cell-based therapy for osseous regeneration.
Established treatments in bone healing disorders
Autologous bone transplantation
Autologous bone transplantation is the therapy of choice for treating bone healing disorders. Despite its high efficiency in regenerating bone tissue, autologous (cancellous) bone transplantation does have numerous disadvantages. These include a longer surgery time, damage by surgical exposure (e.g. subcutaneous nerves), persisting pain and swelling at the donor site, and impaired esthetics due to scar formation or osseous malformation. Furthermore, the potential for osseous regeneration of autologous bone grafts in elderly people is low compared to an increased donor site morbidity in this population.2
The growing interest among experts can also be seen in the increasing number of publications dealing with donor site morbidity().
The increasing frequency of publications on “donor site morbidity” and “bone” listed by Medline reflects the growing interest and examination of bone harvesting-related damage.
New bone formation in long bones is achieved using callus distraction, including the so-called segment transport and external fixation devices. There must be osseous interruption which is fracture-related or created by an osteotomy. Disadvantages include the fact that the process can continue for months, the risk of infections transmitted via the pin tracks of the fixation, and the lack of application possibilities to the pelvis, spine, thorax, skull or to the hand and foot skeletons.
Ultrasound and shock waves
Using extracorporeal shock wave therapy to regenerate bone is mostly restricted to treating atrophic pseudarthrosis. Critical size bone defects cannot be healed by this non-operative therapy.
Biological fundamentals and rationale of cell-based therapy of bone defects and bone healing disorders
The rationale for a cell-based therapy to induce bone tissue regeneration is based on the high osteogenic potency of undifferentiated or almost undifferentiated osteoblastic progenitor cells of various origins. This has been documented in a now vast number of pre-clinical studies.3
For ethical and biological reasons, stem cell therapy on the musculoskeletal system is limited to autologous transplantation of postpartum progenitor cells. Omnipotent (the potential to regenerate a complete, viable organism) or totipotent (potential to regenerate different types of tissue) embryonic stem cells, on the other hand, are used only in experimental studies.
Quantitatively relevant amounts of mesenchymal, multipotent progenitor cells are found not only in human bone marrow, but particularly also in the periosteum and in adipose tissue.4–6
On the other hand, stem cells with osteoblastic potency, occur in lesser quantities in numerous other tissues, such as muscle,7
umbilical cord blood,8
Osteoblastic differentiation of mesenchymal stem cells passes through numerous intermediary stages, whereby it is less the original tissue of the MSC than the local environment with correspondingly different stimuli that influences the kinetics, gene expression and protein synthesis of the cells. The mechanisms of intracellular signal transduction are complex and most clinically oriented orthopedic surgeons can barely grasp the overall picture (). Depending on the degree of differentiation of the osteoblastic precursor, different typical proteins and antigens are expressed in different amounts (). As differentiation increases, the cellular proliferation rate falls.
Figure 2 Summary of important intracellular pathways of signal transduction during osteoblastic differentiation. Cytomechanic stimuli, BMPs and inflammatory stimuli, in particular, encourage osteoblastic differentiation. The expression of some of the listed KO-factors, (more ...)
Other characteristics which make autologous mesenchymal progenitor cells an attractive candidate for the treatment of bone defects are:
- simple availability and an uncomplicated harvesting technique by aspiration without the disadvantage of significant harvesting morbidity;
- the standardized and well-established isolation technique using density gradient centrifugation or flow cytometry technology (e.g. fluorescence activated cell sorter, FACS);
- a consensual definition of the term “mesenchymal stem cells” (MSC) that has now been worked out, with well-defined biological properties17–19 ();
Table 1 Consensual definition of the term “mesenchymal stem cell”. A large number of synonyms exist, however, in scientific literature, e.g. precursors of non-hematopoietic tissue, colony forming units-fibroblasts, marrow stromal cells, bone marrow (more ...)
- the simple cultivation technique for in vitro expansion and determination of the proliferation rate through colony forming units (CFU);
- that osteoblastic differentiation can be well controlled in in vitro cultivation with stimuli such as dexamethasone, ascorbic acid and β-glycerol phosphate (DAG), and the decades of experience with this stimulation method;
- the availability of defined cellular expression markers by which osteoblastic differentiation can be reliably documented (e.g. osteocalcin, osteopontin, osteoprotegerin, Cbf1/Runx2, collagen type I, alkaline phosphatase, osterix, bone sialo protein, signs of biomineralization, RANKL);20
- the immunosuppressive and immunomodulatory effects of MSC which lead to a limitation of local inflammatory reactions at the transplantation site;21
- the good adherence to surfaces, which favors the use of scaffolds and advocates local concentration of in vivo introduced cells at the transplantation site;22
- the decades of experience in bone marrow transplantations in hemato-oncology, which is supported by the low transplantation risk especially in autologous transplantations.
Clinical application of cell therapies in bone healing disorders
In contrast to the extensive in vitro and animal experiment data, there are only a few studies that show clinical results for cell therapy treatments to regenerate bone.
There are two clinical application forms of cell therapies to regenerate bone. Besides the biological differences, various health law-related consequences also emerge for the manufacturer and the orthopedic surgeon in attendance.
- Cell therapies without expansion in culture:
- with the isolation of defined primary cells;
- without the isolation of defined primary cells.
- Cell therapies with ex vivo expansion:
- with typical cell differentiation;
- without typical cell differentiation.
Cell therapies without expansion in culture
What is generally meant here is cell therapies that are harvested or produced during an operation. The tissue used for this does not leave the operation theatre or operation area and is, therefore, under the direct supervision and responsibility of the operator in attendance. Bone marrow aspiration concentrate (BMAC) is a typical example of this form of application. At the beginning of the operation, a defined volume of bone marrow is harvested by Jamshidi-vacuum aspiration of the ventral or dorsal iliac crest and suspended in an anti-coagulating heparin and ACDA solution in a transfusion bag. Mononuclear cells are then isolated from the harvested bone marrow aspirate in a density gradient centrifuge in the closed system that we have been using since 2005.
Possible quality controls of the cell therapy (BMAC) are to compare the number of cells in the BMAC with that in the initial aspirated bone marrow, and determine the CFU-F and ALP activity during in vitro
Despite these quality parameters, the individual potency of in vivo
applied cell therapies cannot be reliably predicted. Some publications indicate, however, that compared to the transplantation of a defined type of cell, applying mixed populations of mesenchymal and hematopoietic progenitor cells at different stages of differentiation is more effective for osteogenic regeneration.26
In a prospective clinical study and in various experimental treatments, our research group has so far successfully treated over 100 patients with local bone healing disorders using a BMAC biomaterial composite. Fifty percent of the bone defects were grafted with autologous cancellous bone and the remaining 50% with a BMAC biomaterial composite (hydroxylapatite, Orthoss®, Geistlich, Wolhusen, Switzerland vs. collagen sponge, Gelaspon®, Chauvin Ankerpharm, Berlin, Germany). So far, our study has found that the use of BMAC reduces the harvest of autogenous bone by 50% with no slowing down or absence of bone healing being observed.27, 28
No complications with the application were observed in any of the patients. The low complication risk of this procedure29
and the osteogenic potency in the parallel application of different biomaterials has also been reported by other research groups.30, 31
A high variance in the number of harvested cells was observed in the human bone marrow aspirate.32, 33
To maximize the yield of bone marrow cells, the following procedure is recommended.
- Create a sufficiently high vacuum: this is necessary to create sufficient local force to retrieve the (in contrast to hematopoietic cells) strongly adherent mesenchymal cells from the tissue mass. If the vacuum is too low, the amount of peripheral blood in the aspirate will be higher. As well as the plunger pressure created by the operator, the geometry of the syringe also plays an important role in creating sufficient negative pressure().
Figure 3 In iliac crest-vacuum aspiration, the geometry of an aspiration syringe influences the proportion of MSCs in the aspirate. The pressure required to retrieve the mesenchymal cells is exerted at the tip of the needle and is defined by the formula: pressure (more ...)
- Draw several small portions in small aspiration volumes: the number of mesenchymal progenitors (and the vacuum) per aspiration volume decreases during an aspiration procedure.34 It is recommended, therefore, that you draw a maximum 5 mL of bone marrow and then create a new vacuum.
- The number of mesenchymal progenitor cells falls with repeated aspiration procedures at the same spot. A maximum 3 aspirations at the same spot before positioning the needle at another spot is, therefore, recommended.
- More cells are retrieved with parallel insertions of the aspiration needle than with diverging insertions in one area().
Figure 4 The areas of the iliac crest reached by the tip of the aspiration needle if inserted at diverging angles at the same point overlap, so that areas that have been perforated and aspirated once already are subjected to the procedure several times. This leads (more ...)
Also ensure sufficient anti-coagulation of the bone marrow aspirate during the harvesting procedure. Heparin and ACDA solutions are used for this. The aspiration needles and syringes should be flushed with the solution before use. Density gradient centrifugation is particularly suitable for isolating mononuclear cells for bone regeneration therapy.23
Cell therapy treatments with temporary ex vivo expansion
In orthopedics and traumatology, autologous cell therapies have been used regularly on the musculoskeletal system after ex vivo
cultivation, at least since the clinical introduction of autologous chondrocyte transplantation (ACI). Unlike cartilage regeneration, for which ACI was used in more than 12,000 patients between 1987 and 2005,35, 36
there are no reliable data on osseous regeneration after temporary in vitro
cultivation. In the treatment of necrosis of the femoral head, for instance, whereas numerous one-step transplantations are documented, only three case studies with a maximal observation period of three months can be found. Here, a mixed cell population from bone marrow cells (so-called tissue repair cells, TRCs), was expanded over 12 days under GMP conditions and then transplanted autologously together with a scaffold made of tricalcium phosphate (TCP) within the framework of core decompression.37
The particular drawbacks of temporary cultivation of MSCs lie not only in the considerable logistical effort to ensure the quality of the cell therapy treatment but especially in the biological characteristics of this cell population. As soon as MSCs are isolated from their tissue mass and transferred to a culture dish, differentiation proceeds in accordance with the culture conditions.38–40
The yet inconclusive biological effects when fetal bovine serum is used in the culture, as well as telomere shortening, and thus cell aging with ex vivo
cultivation also have to be considered. Furthermore, analysis of 170 neoplasia-associated DNA promoters was able to show that despite the relatively high genetic stability of MSCs from human bone marrow or adipose tissue, damage in the genome could occur at later stages.41
The question as to whether these genotoxic effects of prolonged in vitro
cultivation are also clinically manifested after re-transplantation remains unanswered, however. The potential effects of changes in the chromatin structure due to epigenetic factors at the beginning of osteoblastic differentiation also remain largely unknown.42
Cell therapy in local bone defects, bone healing disorders and osteonecrosis
Other research groups have also reported positive clinical results after using human bone marrow cells. Giannini et al.
showed that in patients with osteochondral defects in the talus, functional improvements were achieved through autologous bone marrow cell transplantation by arthroscopic surgery.43
As early as 1991, Conolly et al.44
reported equivalent healing rates for autologous bone marrow grafting to treat post-traumatic pseudarthrosis
of the tibia. Other authors also support the high osseous regeneration potency of the percutaneous implantation of autologous bone marrow concentrate to treat pseudarthrosis32, 33
and discuss supplementary osteoblastic stimulation using platelet rich plasma (PRP).45
Some authors, on the other hand, have reason to believe that bone regeneration through cell therapy also depends very much on the transplantation site and the local blood supply. According to a study by Kitho et al.
, in which ex vivo
-cultivated MSCs were used together with PRP in 51 lengthening osteotomies, cell therapy accelerated bone healing in the femur compared to in the tibia.46
Overall, however, the cell therapy showed no advantages over the untreated control group and, moreover, no relation between the bone healing rate and the number of transplanted cells or the PRP concentration was found. Hernigou et al.
, however, reported that grafting over 50,000 osteoblastic progenitor cells particularly encouraged healing in atrophic tibial pseudarthrosis.33
Minimally invasive cell therapy of solitary or aneuryamal bone cysts
via percutaneous implantation of autologous bone marrow is favored by a number of authors owing to the healing rate of over 80% 47, 48
(). On the other hand, a randomized clinical study showed that in the treatment of simple bone cysts, autologous bone marrow injections were inferior to local steroid injections.49
Figure 5 Healing course after autologous cell therapy with bone marrow aspiration concentrate (“BMAC”) augmented with HA granules in a 4-year old male patient with a large aneurysmal bone cyst of the proximal femur. Ten months after surgery, relevant (more ...)
Initial results are also available on cell therapy treatments to promote osseous fusion at the spine
. After an observation period of 34 months, fusion rates of over 90% were found for tricalcium phosphate (TCP)-bone marrow composite transplantations.50
For an HA-collagen-I composite incubated with autologous bone marrow, the posterolateral lumbal fusion rate was found to be the same as for autologous bone transplantation, but fusion rates were not the same for intracorporeal fusions.51
Cell therapies have been used successfully to treat avascular osteonecrosis
(AVN) for many years by Hernigou et al.52, 53
and, in the meantime, also by other research teams in experimental treatments and clinical studies.54–56
Patients with sickle cell anemia-related AVN, in particular, benefit from a local injection of mononuclear bone marrow cells. Patients with steroid-related AVN have a worse prognosis when treated with MSCs to regenerate bone.57
The number of implanted cells and their proliferation potency, as measured by the CFU-F, are positive predictors for successful bone marrow concentrate therapy in the treatment of osteonecrosis.52
It is unclear whether the reduced number of MSCs in the proximal femur observed in patients with AVN is an independent risk factor in the development of an AVN, or is resulting from AVN.57
Other authors, however, report comparatively high numbers of osteoblasts in the major trochanter region with necrosis of the femoral head.58
It has been shown that for ARCO stages I and II local cell therapy with autologous bone marrow in combination with core decompression diminished the risk of a medium-term progression of necrosis of the femoral head.59
A multi-center study on patients with peripheral artery disease (PAD) amply documented that mixed cell populations from human bone marrow not only have an osteoblastic but also an angiogenetic effect.60
Because of the lack of control groups, however, cell therapy is mentioned in the current S1-recommended treatments of atraumatic necrosis of the femoral head in adults under “Operation methods without good documentation support
Besides direct transplantation of the cell suspension into the AVN area within the framework of a core decompression, a cancellous bone graft can also be combined with the autologous cell therapy.62
For this, a Krohn hollow mill is used to extract a cylinder of cancellous bone and the AVN area subjected to curettage through the resulting cavity. After removing the macroscopic avascular tissue for histopathological diagnosis, the cylinder of cancellous bone is incubated with the cell therapy and then re-implanted in the osseous defect. Medium- and long-term results of this cell therapy treatment are pending. is a summary of the results of clinical applications of cell therapies to regenerate bone.
Table 2 A summary of published studies including more than 10 patients after cell therapy in bone defects or bone healing disorders. The medline review showed that cell therapy by bone marrow is not a new technique but has been applied for almost 20 years. According (more ...)
Cell therapy in skeletal diseases
In addition to the local application of MSCs, data are also available for MSCs in the successful treatment of skeletal diseases which are accompanied with deficiencies in the bone structure.78, 79
In 1999, after numerous in vitro
experiments and animal experimental studies, Horwitz et al.
, for the first time, treated 3 children with osteogenesis imperfecta
(OI) with allogenic transplantations of mesenchymal bone marrow cells.80
The cells were introduced intravenously after ablative pre-treatment of the patient (chemotherapy and immunosuppression). Post-operative bone biopsies after 216 days and bone density measurements showed a significant quantitative and qualitative improvement in the bone structure. In another publication from the year 2001, the same research group81
reported their findings on 5 OI patients who had been treated with cell therapy and on 2 other OI patients without cell therapy treatment (OI type III). After an investigation period of six months, children who had received cell-based therapy showed an accelerated growth rate.
is another skeletal disease involving insufficient osteoclast activity that is currently being treated with autologous bone marrow transplantation. Driessen et al.82
found that the probability of 5-year disease free survival was 73% after cell-based therapy. Treatment before the age of three years improved the chances of success of cell therapy in osteopetrosis.83
However, due to the severe side-effects and possible complications (e.g. graft rejection, hypercalcemic crises, pulmonary hypertension, delayed hematopoiesis, veno-occlusive disease), allogeneic cell therapy treatment of patients with osteopetrosis is limited to severe manifestation of disease.84
Three case reports also report the successful treatment of an 8-month old infant with infantile hypophosphatasia
who underwent transplantation of T-cell depleted bone marrow from the sister. The positive effects of the cell therapy ceased, however, after six months. Twenty-one months after the first transplantation, a second transplantation of ex vivo
expanded bone marrow cells took place resulting in an increase in bone mass. At the age of six, the patient in question still showed signs of stunted growth but displayed normal intelligence.85
Another approach in cell therapy treatment of infantile hypophosphatasia consists of intra-peritoneal, subcutaneous or intraosseous bone transplantation from a related donor parallel to the intravenous bone marrow injection. The postulate here is that migration of the donor MSCs in the recipient organism will positively influence bone healing and the rejection reactions.86, 87
It is unclear whether cell-based therapy will also gain acceptance in other skeletal diseases, such as osteoporosis.
Outlook for the future
Due to the accelerated aging of osteoblastic progenitor cells after in vitro
cultivation, the limited resources, the diminished osteoblastic potency with increasing age and the improved standardizations, immortalized human MSCs are currently undergoing pre-clinical investigations for their suitability for cell therapy.88, 89
One way of avoiding aging of MSCs is to transfer the cDNA of telomerase reverse transcriptase (hTERT). With this enzyme, the telomeres that have been shortened during the course of replication are returned to their original length. Some authors were able to demonstrate a high osteoblastic potency in vitro
and in animal experiments with this process.88
Nevertheless, in view of the current legislation, it is uncertain whether these new therapy procedures can also be tested clinically. In addition to cell-based therapies, there are other innovative “orthobiologics” with bone regeneration as the goal. These include anchor proteins that stimulate osteoblastic adherence, e.g. RGD sequences, fibronectin or peptide 15. α-granules of thrombocytes, in particular, contain large amounts of growth factors with osteoblastic and cell proliferation potency, such as transforming growth factor-β (TGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).90
Whether it makes sense and is necessary, within the context of the clinical application of cell therapies, to increase the osseous potency of osteoblastic progenitor cells using additional growth factors must first be investigated in controlled clinical studies given that the hitherto existing data are contradictory and not sufficiently reliable.46,91,92