Intraoperative Construct Preparation: A Practical Route for Cell-Based Bone Regeneration

Abstract

Stem cell-based bone tissue engineering based on the combination of a scaffold and expanded autologous mesenchymal stem cells (MSCs) represents the current state-of-the-art treatment for bone defects and fractures. However, the procedure of such construct preparation requires extensive ex vivo manipulation of patient's cells to achieve enough stem cells. Therefore, it is impractical and not cost-effective compared to other therapeutic interventions. For these reasons, a more practical strategy circumventing any ex vivo manipulation and an additional surgery for the patient would be advantageous. Intraoperative concept-based bone tissue engineering, where constructs are prepared with easily accessible autologous cells within the same surgical procedure, allows for such a simplification. In this study, we discuss the concept of intraoperative construct preparation for bone tissue engineering and summarize the available cellular options for intraoperative preparation. Furthermore, we propose methods to prepare intraoperative constructs, and review data of currently available preclinical and clinical studies using intraoperatively prepared constructs for bone regenerative applications. We identify several obstacles hampering the application of this emerging approach and highlight perspectives of technological innovations to advance the future developments of intraoperative construct preparation.

Introduction

Large bone defects and fractures are still clinical problems that require surgical intervention, especially in a condition where the regenerative process is compromised.¹ Although autografts are currently considered the gold standard for such treatment, their application is restricted by limited supply and associated complications.² Allografts from cadaveric bone and xenografts from nonhuman species are considered as another alternative for reconstructing bone defects, but are restricted by immunological rejection and disease transmission issues.^3,4 Attempts to find superior substitutes for bone grafts have resulted in the emergence of bone constructs based on tissue engineering principles. The term ‘bone tissue engineering’ defines the development of biologic bone graft substitutes with a synthetic scaffold, osteogenic cells or growth factors to repair damaged, or regenerate new bone.⁵ In particular, cell-based bone tissue engineering has gained much attention due to vast investigation of stem cells and controlled bone regeneration over growth factor-based strategies.

The classical procedure of cell-based bone tissue engineering consists of isolating mesenchymal stem cells (MSCs) from a patient, expansion and differentiation in culture, and seeding onto a suitable scaffold before implantation back into the same patient⁶ (Fig. 1a). MSCs from different sources, such as bone marrow (BM), adipose tissue, peripheral blood (PB), and dental pulp, have been explored extensively and have demonstrated favorable bone regenerative capacity in many animal studies (reviewed by Ma et al.).⁷ Furthermore, several clinical trials with cell-based constructs for bone regeneration have been conducted and also showed feasibility and efficacy regarding bone formation (reviewed by Grayson et al.).⁸ These convincing outcomes have provided a solid foundation for further use of cell-based bone tissue engineering for bone repair. In addition, vast scientific and technical input have reduced many practical and regulatory barriers by optimizing the preparation procedure and the outcome of tissue-engineered bone constructs and by investigating the mechanism of cell-based bone repair. For instance, the risk of using animal serum for MSCs culture has been (partly) solved by using human platelet lysate as an alternative.^9,10

FIG. 1.

The schematic representation of (a) conventional bone tissue engineering and (b) intraoperative concept-based bone tissue engineering. In the conventional bone tissue engineering, cells are isolated from a biopsy, expanded in monolayer culture, seeded onto a 3D scaffold and cultured in vitro for a certain period. The engineered graft is finally implanted into the patient. This procedure generally takes from 1 to 3 weeks. In contrast, the intraoperative concept-based tissue-engineered bone constructs circumvents the monoculture and expansion procedure by isolating cells from harvested tissue and then preparing a construct during the same surgery. The whole procedure can be finalized in several hours. Color images available online at www.liebertpub.com/teb

Despite these great advances, several limitations of bone tissue engineering have been brought forward, such as the time-consuming and costly in vitro expansion procedure (Fig. 1a), the risk of pathogenic contamination and safety concerns of genetic alterations during cell passages, and high cost and patient inconvenience from two invasive surgical procedures. These problems hinder the practical application of bone tissue engineering and encourage researchers to prepare tissue-engineered bone constructs in one-step surgical procedure without cumbersome in vitro manipulation. We defined this approach as intraoperative preparation of tissue-engineered bone constructs. The intraoperative preparation of tissue-engineered bone constructs indicates that the manufacturing process, including harvest of autologous cells, generation of constructs, and finally implantation, can be combined with the surgical procedure to treat bone defects (Fig. 1b). This method entails several benefits aiming at the limitations of conventional bone tissue engineering strategies: (1) without in vitro cell manipulation and expansion procedure, surgery can be performed within several hours, which would dramatically reduce cost and labor and is more patient-friendly; (2) without using exogenous components such as animal serum for several passages expansion, the risk of gene mutation and pathogenic contamination likely becomes negligible; (3) due to the manipulation and surgery performed entirely within one operating room and the minimal manipulation of cells or tissues, this approach would ease the route to regulatory approval.

In the following sections, we review current approaches to intraoperatively prepare tissue-engineered constructs, with which cells can be harvested, minimally manipulated, and implanted to bone defect areas during surgery. Based on the reviewed articles, we here discuss cells that are intraoperatively available to prepare such constructs and provide an overview of available data of preclinical and clinical studies using intraoperatively prepared tissue-engineered bone constructs. Furthermore, the therapeutic efficiency and future perspectives and technologies to develop such intraoperatively prepared tissue-engineered bone constructs are discussed.

Available Cell Sources

Osteogenic cells are the most essential component to intraoperatively prepare tissue-engineered bone constructs with high osteogenic activity. Although cells with osteogenic potential are present in many tissues, to meet the requirements for intraoperative preparation, only a few tissues have been explored, namely, BM, adipose tissue, and PB. The advantages and disadvantages of utilization of these tissues for intraoperative construct preparation are displayed in Table 1.

Table 1.

The Advantages and Disadvantages of Using Easily Available Cells from Different Tissues to Make Intraoperative Concept-Based Bone Constructs

Cell source	Cell type	Advantages	Disadvantages
Bone marrow	BMACs	High osteogenic potential	Donor variation^85–87
	BMACs	Endothelial subpopulation	No minimally invasive harvest
	BMNCs	High osteogenic potential	Low percentage of MSCs^19,88
	BMNCs	Extensively studied	No minimally invasive harvest
Adipose tissue	SVF	Abundant tissue	Donor variation^31,90
		Easy harvest procedure	Lower osteogenic potential^31,89
		Endothelial subpopulation^89,90	Lower osteogenic potential^31,89
	PSCs	High yield from adipose tissue	Multistep isolation procedure
	PSCs	Homogenous cell population	Loss of endothelial subpopulation
Peripheral blood	PBMNCs	Noninvasive harvest procedure	Low percentage of MSCs^37,42,91
	PBMNCs	Endothelial subpopulation⁴⁴	Presence of undesired cells
	PB-derived EPCs/ECs	Noninvasive harvest procedure	Low yield⁴⁷
	PB-derived EPCs/ECs	Easy isolation method	Not widely studied

BMACs, bone marrow aspiration concentrate cells; BMNCs, bone marrow-derived mononuclear cells; SVF, stromal vascular fraction; PSCs, perivascular stem cells; PBMNCs, peripheral blood mononuclear cells; PB, peripheral blood; EPCs, endothelial progenitor cells; ECs, endothelial cells; MSCs, mesenchymal stem cells.

Bone marrow

BM is to date the most common tissue source applied both experimentally and clinically for preparing intraoperative tissue-engineered bone constructs, mainly relating to its high osteogenic capacity and relatively easy access. The first developed strategy was the direct use of bone marrow aspiration concentrate cells (BMACs),^11,12 which are a mixture of erythrocytes, granulocytes, hematopoietic cells (HCs), endothelial progenitor cells (EPCs), MSCs, lymphocytes, and immature monocytes among others. This approach can be performed in a short time (∼30 min) and at relatively low cost. Approximately 2 × 10⁷ BMACs per ml of BM can be simply obtained using a reamer-irrigator-aspirator and counted using a hemocytometer.¹³ Although a few studies reported positive effects of using these cells for bone regeneration,^14–16 the majority of studies involving BMACs demonstrated a lack of stimulatory effects on bone regeneration,^11,17,18 which can likely be attributed to the relatively low numbers of osteogenic cells and a high number of noneffective cells.

After aspiration of BM and elimination of erythrocytes and granulocytes, a heterogeneous cell population, that is, bone marrow-derived mononuclear cells (BMNCs), can be achieved. This fraction includes a higher number of HCs (around 4%), MSCs (around 0.1%), and EPCs (around 5%). Multiple animal studies have demonstrated beneficial effects of BMNCs on bone healing.^17,19–21 A large-scale clinical report also demonstrated the safety and efficacy of BMNCs for intraoperatively prepared tissue-engineered bone constructs.²² However, due to the limited volume of BM that can be used for implantation in one patient (maximal ∼300 mL) and the relatively low yield of MSCs in BM (maximum 100 colony-forming unit fibroblasts [CFU-f]/million MNCs in young people), only 1500–3000 MSCs/mL in healthy human BM can be obtained,²³ which cannot provide the desired or required numbers of MSCs, especially in elderly or otherwise osteogenesis-compromised (e.g., osteoporotic) patients.

Adipose tissue

In contrast to the limited supply of BM, adipose tissue is largely available and has recently attracted much attention as a promising alternative source for osteogenic cells. Fat tissue can be collected through a less invasive method with minimal morbidity upon harvest and in larger quantities of effective stem cells than BM. The numbers of MSCs in adipose tissue (i.e., 1–3 × 10⁴ CFU-f/million MNC) is 100–300 times higher compared to BM,²⁴ and the number of stem cells that can be isolated per unit volume of lipoaspirate is ∼10 fold greater than that from BM (i.e., 3 × 10⁴ MSCs/mL of lipoaspirate). Consequently, small adipose tissue reservoirs can already provide sufficient numbers of MSCs for clinical applications. In addition, perivascular stem cells (PSCs), another stem cell type that is abundant in fat tissue,²⁵ have been demonstrated to have a mesenchymal potential equal or even superior to conventional MSCs in some cases.^26,27 James et al. demonstrated the efficacy of using PSCs for intraoperative preparation of tissue-engineered bone constructs.²⁵

Stromal vascular fraction (SVF), the freshly isolated fraction from the lipoaspirate or fat patch, is a mixture of several cell types, including MSCs, EPCs, pericytes, and monocytes.²⁸ Since the isolation of SVF can be achieved within a few hours (maximum 4 h in available reports),^29–31 it is suitable for a one-step surgical procedure to prepare tissue-engineered bone constructs. Another key advantage of SVF is the presence of endothelial lineage cells among the heterogeneous cell mixture. These cells, which are typically lost during prolonged monolayer cultures, but preserved upon direct construct preparation, have shown to contribute to the formation of blood vessels in recipient sites, which is critical for the survival and function of cells in implanted tissue-engineered bone constructs.³² Nevertheless, a few studies have postulated concerns that the osteogenic capacity of SVF is significantly lower than that of MSCs isolated from BM.^33,34

Peripheral blood

As blood is more easily accessible than BM and adipose tissue, PB is emerging as a source of MSCs. Mononuclear cells (MNCs) from PB contain both hematopoietic stem cells and EPCs.³⁵ The osteogenic potential of PB-derived MSCs (PBMSCs) has been suggested by accumulating evidence from both in vitro and in vivo experiments.^36,37 However, only small numbers of stem cells exist in PB (less than 20 CFU-f/million MNCs) compared to numbers in BM,³⁸ which is a major obstacle for an intraoperative approach using cells derived from PB. Several steps have been taken to enrich stem cells in PB, such as mobilizing them from BM with granulocyte colony-stimulating factor,^39,40 chemotherapeutic agents,⁴¹ or using CD133⁺ selection.⁴² This can concentrate PBMSCs over 20 times (to reach levels comparable to those in fresh BM). However, this CFU-f value is still far from the effective implantation threshold (≥1000 cells/cm³ for BMSCs).⁴³ Therefore, the application of PBMSCs for intraoperatively prepared tissue-engineered bone constructs has not been reported to date. Other cell types, for example, CD31⁺ EPCs⁴⁴ and CD34⁺ endothelial/hematopoietic progenitor cells,⁴⁵ are present in large quantities in PB, independent of the individual's age and gender. When applied to rat femoral bone fractures, advanced bone tissue restoration was observed.^44,45 Given its availability (between 70% and 80%) and general leukocyte enrichment efficiency (4 × 10⁶ cells/mL), 5 mL of PB would provide 1 × 10⁷ CD31⁺ cells, which is sufficient to prepare tissue-engineered bone constructs without expansion cultures.

Cell Isolation

Isolation or concentration of desired cells is needed to prepare tissue-engineered bone constructs. This step can select a certain cell mixture or even a specific type of cells and remove detrimental cells. For BM-derived cells, density separation (DS) and selective retention (SR) are the mostly adapted methods for isolating desired cells. DS involves the use of a centrifuge to concentrate nucleated cells and connective tissue progenitors, while SR involves adsorbing connective tissue progenitor cells through a porous substrate. Recently, a method based on red cell lysis was established to concentrate BMNCs over 50 times.¹⁴ This method is more efficient, faster, and more easily standardized compared to DS and SR, showing promise in clinical applications for preparing BM-derived intraoperative bone constructs.

To obtain SVF for intraoperative preparation of tissue-engineered bone constructs, adipose tissue is generally harvested via minimally invasive techniques, and processed by enzymatic digestion and centrifugal enrichment. To meet the requirement from regulatory authorities for “less than minimally manipulated” cells, a nonenzymatic method was recently developed to obtain highly enriched adipose tissue-derived elements by mild mechanical forces,⁴⁶ showing another option to intraoperatively obtain desired cells from lipoaspirate. To further select a subpopulation from SVF, such as CD31⁺ or CD34⁺ cells, cell sorting by flow cytometry (FACS) is mostly used based on the surface markers of desired cells. Due to the special format of PB, isolation of blood-derived cells is generally performed by concentrating the buffy coat first and then sorting desired cells with magnetic technique⁴⁷ or FACS.^48,49

Fabrication of Scaffold

A scaffold is an essential part of intraoperative preparation of tissue-engineered constructs to provide (i) a bed for cell seeding, (ii) mechanical support during regeneration, and (iii) ingrowth of host cells during regeneration. Although various materials, including bioceramics, hydrogels, polymers, and natural bone-derived materials, are used in preclinical studies for intraoperative construct preparation, the “gold standard” scaffold in clinical studies is still auto-/allo-/xenografts and few synthetic bioceramics. This probably relates to their suitable mechanical and biological properties. Although the crucial effect of scaffold properties on cell attachment and bone formation has been reported in studies using intraoperatively prepared tissue-engineered constructs,^29,31,50 a comprehensive therapeutic evaluation of these commonly used scaffolds for bone repair is still missing.

Except for the use of natural bone grafts and some synthetic bioceramic scaffolds, certain types of patient autologous materials are also utilized during surgery to improve the cell-loading efficiency and therapeutic effectiveness. For example, BM or blood will clot several hours after aspiration to form an autologous fibrin scaffold, which provides cells with a natural environment after seeding.^17,44 This transformation from liquid to solid also benefits the preparation of constructs to match the shape and dimensions of the defect to be repaired. Further, this platelet-rich fibrin gel is able to stimulate migration of host progenitor cells as well as the proliferation and osteogenic differentiation of seeded cells owing to the release of growth factors, including platelet-derived growth factor, transforming growth factor-β, fibroblast growth factor-2, vascular endothelial growth factor (VEGF), and insulin-like growth factor.⁵¹ These ideas have been applied to multiple clinical trials and showed that fibrin suffices as a scaffold enabling implanted cells to remain at the fracture site and to function synergistically with released growth factors.^30,52–56

Preclinical Studies with Intraoperatively Prepared Tissue-Engineered Bone Constructs

Following a general concise review screening procedure, 34 preclinical studies were identified (Table 2): 7 animal studies using ectopic implantation models and 27 animal studies using an orthotopic model. Among these preclinical studies, 12 studies used BM-derived cells, 19 studies used adipose tissue-derived cells, and 4 studies used PB-derived cells to regenerate bone with an intraoperative preparation concept (Fig. 2). One study utilized cells from different sources (i.e., adipose tissue and BM) to evaluate their bone healing capacity.

FIG. 2.

The pie charts showing the number of reported studies using easily accessible cells from different tissues for intraoperative concept-based bone tissue engineering in preclinical and clinical studies. Cells from bone marrow, adipose tissue and peripheral blood were used for intraoperative concept-based bone tissue engineering in preclinical and clinical studies. Adipose tissue-derived cells dominated the preclinical studies involving intraoperative preparation of tissue-engineered bone constructs. In contrast, bone marrow-derived cells were the main source for usage in clinical trials. Color images available online at www.liebertpub.com/teb

Table 2.

Preclinical Studies with Intraoperatively Prepared Tissue-Engineered Bone Constructs

Cell source	Cell isolation/treatment	Scaffold	Seeding density	In vivo time	Animal model	Evaluation method	Efficacy of bone formation	Reference
Ectopic studies
Rat BMACs	Aspirate	HA/TCP	Unclear	8 weeks	Rat subcutaneous	Histology	Negligible osseous tissue	¹¹
Murine SVF	Centrifuge	PRP, BMM	5 × 10⁴/cm³	8 weeks	Mouse subcutaneous	Histology	No bone formation	⁹²
Human SVF	Centrifuge	PRP, β-TCP, HA, Bio-Oss	2 × 10⁶/cm³	8 weeks	Nude mouse subcutaneous	Histology	No frank bone tissue	³¹
Human SVF	Centrifuge	PRP, HA	1, 4 × 10⁶/construct	8 weeks	Nude mouse subcutaneous	Fluorescence microscopy	No frank bone tissue	⁹³
Human SVF	Centrifuge	PRP, allogenic cartilage	0.24–1 × 10⁶/construct	12 weeks	Nude mouse subcutaneous	μCT, histology	SVF (5 × 10⁵/construct) formed more bone than cell-free and other cell density constructs	⁶⁹
Human SVF, PSCs	FACS	DBM	2.5 × 10⁵/construct	4 weeks	SCID mouse intramuscular	μCT, histomorphometry	PSC formed more bone than SVF	²⁵
Human PSCs	FACS	DBM	2.5 × 10⁵/construct	4 weeks	SCID mouse intramuscular	μCT, histomorphometry	PSC formed more bone than cell-free control	⁹⁴
Orthotopic studies
Rat BMACs	Aspirate	HA/TCP	Unclear	8 weeks	Rat femoral segmental defect	Histomorphometry	No difference between BMACs and cell-free control	¹¹
Canine BMACs, BMNCs	SR	DBM	122, 269 × 10⁶/construct	12 weeks	Canine spinal fusion	CT	Enriched BMNCs had greater fusion volume and area than BMACs and cell-free	⁶⁴
Canine BMACs, BMNCs	SR	PRP, DBM	Unclear	16 weeks	Canine segmental femoral defect	Histology	BMNC/PRP healed a higher percentage of bone defects than BMACs and cell-free	¹⁷
Canine BMACs, BMNCs	DS, SR, DS + PRP	Allografts	58, 110, 76, 276 × 10⁶/construct	4 weeks	Canine femoral multidefect model	μCT	No difference in bone formation between BMACs and DS-BMNCs, SR-BMNCs and DS+PRP-BMNCs	¹⁵
Canine BMACs, BMNCs	Red blood cell lysis	β-TCP	3.1, 169.3 × 10⁶/construct	24 weeks	Canine orbital bone defect	Histology	BMNCs had more bone formation than BMACs and cell-free	¹⁴
Canine BMACs, BMNCs	Magnetic sorting	Allografts	16.1, 20.5 × 10⁶/construct	4 weeks	Canine femoral defect	Histomorphometry	BMNCs had more bone formation than BMACs	²⁰
Ovine BMACs, BMNCs	SR	β-TCP	Unclear	6 months	Ovine spinal fusion	μCT, histology	Denser bone formation in the BMNCs than BMACs and cell-free	¹⁸
Ovine BMACs, BMNCs	FICOLL centrifuge	PRP, BMM	5.34, 44.5 × 10⁴/construct	16 weeks	Ovine sinus augmentation	Histomorphometry	BMNCs formed more bone than BMACs	⁷⁴
Canine BMNCs	SR	Cancellous BMM	Unclear	12 weeks	Canine spinal fusion	CT	BMNCs formed more bone than cell-free control	¹²
Human BMNCs	FICOLL centrifuge	β-TCP	1 × 10⁶/defect	8 weeks	Nude rat femoral defect	Histomorphometry	BMNCs formed more bone than cell-free	⁹⁵
Murine SVF, PSCs	Magnetic sorting	Allografts	1 × 10⁶/construct	12 weeks	Murine segmental femoral defect	μCT	PSCs based constructs formed more bone than cell-free	⁹⁶
Rat BMNCs, SVF	Centrifuge	BCP	2.5, 5 × 10⁶/construct	3 weeks	Rat irradiated tibial and femoral defect	SEM	BMNCs had more bone formation than SVF, no difference between SVF and cell-free	⁶²
Rat SVF	Centrifuge	PRP, DBM, DBM/PLA	1 × 10⁵/construct	8 weeks	Rat calvarial defect	Radiodensitometry	SVF showed more abundant bone formation than cell-free	⁷⁰
Rat SVF	Centrifuge	Type I collagen gel	1 × 10⁶/defect	6 weeks	Rat femoral callus distraction	Radiography	SVF had more bone formation compared to cell-free	⁶⁷
Rat SVF	Centrifuge	Chitosan	2.19 × 10⁷/construct	28 days	Rat mandibular defect	Histomorphometry	SVF formed more bone than cell-free	⁶⁸
Rabbit SVF	Centrifuge	PLGA	1 × 10⁶/construct	8 weeks	Rabbit ulna defect	μCT	SVF formed more bone than cell-free	⁶⁶
Goat SVF	Centrifuge	PLCL	5 × 10⁶/construct	6 months	Goat spinal fusion	Radiography	No difference between SVF and cell-free	⁷³
Goat SVF	Centrifuge	PLCL	5 × 10⁶/construct	6 months	Goat spinal fusion	Radiography	SVF showed more spinal fusion than cell-free	⁷¹
Equine SVF	Centrifuge	Collagen sponge	5 × 10⁵/construct	12 weeks	Nude rat calvarial defect	μCT	No difference between SVF and cell-free	⁷²
Human SVF	Centrifuge	PRP, Silicated-HA	8 × 10⁶/construct	8 weeks	Nude rat segmental femoral defect	Histomorphometry	SVF formed more bone than cell-free	³⁰
Human SVF	Centrifuge	PRP, allogenic cartilage	0.24–1 × 10⁶/construct	4 weeks	Nude rat calvarial defect	Histology	SVF formed more bone than cell-free	⁶⁹
Human SVF, PSCs	FACS	PLGA	2.5 × 10⁵/construct	8 weeks	SCID mouse calvarial defect	μCT	PSC formed more bone than SVF, no difference between SVF and cell-free	⁶⁵
Human PSCs	FACS	DBM	0.15, 0.5, 1.5 × 10⁶/construct	4 weeks	Nude rat spinal fusion	μCT, histomorphometry	PSC formed more bone than cell-free	⁶³
Rat PBMNCs, PB-derived EPCs	Magnetic sorting	Autologous blood clot	Unclear	6 weeks	Rat femoral defect	μCT, histomorphometry	EPCs advanced bone tissue restoration than PBMNCs	⁴⁴
Human PBMNCs	FACS	Atelocollagen	1 × 10⁶, 1 × 10⁷/construct	8 weeks	Nude rat femoral nonunion	Histology	10⁷ group showed superior bone healing than 10⁶ and cell-free, no difference between 10⁶ and cell-free	⁴⁸
Human PB-derived EPCs	FACS	Atelocollagen	1 × 10³, 10⁴, 10⁵/construct	8 weeks	Nude rat femoral defect	Radiography	More fracture healing in 10⁵ and 10⁴ group than 10³ and cell-free group, no difference between 10³ and cell-free	⁴⁹
Human PBMNCs, PB-derived EPCs	FACS	Atelocollagen	1 × 10⁵, 1 × 10⁷/construct	12 weeks	Nude rat femoral nonunion	Radiography	EPCs formed more bone than PBMNCs and cell-free	⁴⁵

HA, hydroxyapatite; β-TCP, β-tricalcium phosphate; PRP, platelet-rich plasma; BMM, bone mineral matrix; DBM, demineralized bone matrix; BCP, biphasic calcium phosphate; PLA, Poly(lactic acid); PLCL, Poly (L-lactide co ɛ-caprolactone); PLGA, Poly(Lactide-co-Glycolide); DS, density separation; SR, selective retention; FACS, flow cytometry; CT, computed tomography; μCT, micro-computed tomography.

To prepare tissue-engineered bone constructs, normal tissue aspiration combined with either SR, a centrifugation step, or cell sorting (e.g., FACS or magnetic sorting) is the common approach to obtain “easily accessible cells.” To support these intraoperatively isolated cells, different scaffolds were utilized, including allografts, xenografts, collagen, synthetic bioceramics, and polymers. In addition, platelet-rich plasma (PRP), which can be intraoperatively obtained, was used either as a “glue” to entrap cells or as an inducer in eight studies. The effect of PRP addition on bone formation, however, was investigated in none of these studies.

Regarding the mode of action of these intraoperatively prepared tissue-engineered bone constructs, there are a few possibilities. Bone regeneration could be established through direct differentiation of intraoperatively grafted cells.^48,49 Also, one study indicated that potential paracrine effects, including modulatory effects on vascularization, osteogenesis, and inflammatory responses of host tissue, play a more important role than the intrinsic cell differentiation potential.⁴⁴ It is also possible that both mechanisms act simultaneously based on the type of cells and the conditions in which cells are grafted. For instance, when PB-derived CD34⁺ cell-based bone constructs were applied to femoral fractures, direct vasculogenesis and osteogenesis by transplanted PB-derived CD34⁺ cells were detected. Moreover, grafted cells significantly enhanced the intrinsic angiogenesis and osteogenesis of the recipient cells by upregulating VEGF, Angiopoietin-1, and bone morphogenetic protein-2 gene expression at the fracture sites.^48,49 Therefore, it seems likely that these “easily accessible cells” act using both direct and indirect mechanisms to aid in bone healing by promoting neovascularization and osteogenesis.

Clinical Studies with Intraoperatively Prepared Tissue-Engineered Bone Constructs

Among the 30 clinical studies (Table 3 and Fig. 2), 22 publications dealt with cells obtained from BM, 7 from adipose tissue, and 1 from PB. BM-derived cells, especially enriched BMNCs, dominated the clinical trials involving intraoperative preparation of tissue-engineered bone constructs. This is different from what was reviewed for preclinical studies, where adipose tissue was the main source for usage (Fig. 2). This discrepancy probably results from differences in the moment of discovery of the presence of adult stem cells in these tissues and the concomitant amount of (in vitro and preclinical) evidence regarding the bone regenerative potential of isolated cells from these tissues.

Table 3.

Clinical Studies with Intraoperatively Prepared Tissue-Engineered Bone Constructs

Cell source	Cell isolation	Cell treatment	Scaffold	Number of patients	Repair site	Evaluation method(s)	Reported outcome	Reference
BMACs	Aspirate		Allograft	13	Aneurysmal bone cyst	Radiography	Healing obtained in 11 patients after a mean follow-up duration of 3.9 years	¹⁶
BMACs	Aspirate		Local injection	11	Distal tibial metaphysis nonunion	CT	9/11 patients attained bony union within 6 months of bone marrow injection, 6/9 patients reported improved bone healing.	⁸⁶
BMACs	Aspirate		Allograft, β-TCP	5	Sinus floor augmentation	Histology	Graft sites healed for 4–7 months; bone regeneration depended on graft materials	⁸⁵
BMACs, BMNCs	Aspirate DS (device)		Local injection	60	Femur osteonecrosis	MRI	Both methods improved hip function, BMNCs was superior to BMACs to treat avascular necrosis of femoral head.	⁹⁷
BMACs, BMNCs	DS in FICOLL (device)	PRP	Xenograft	11	Maxillary sinus augmentation	Histomorphometry	Biopsies obtained after 3 months indicated no difference in new bone formation between BMACs and BMNCs treated sinus	⁹⁸
BMACs, BMNCs	DS in FICOLL (device)	PRP	Xenograft	11	Maxillary sinus augmentation	Clinical evaluation	Implant survival was not different between BMACs and BMNCs loaded grafts up to 2.5 years after prosthetic loading	⁹⁹
BMNCs	DS (device)		Local injection	116	Osteonecrotic femoral head	Radiography MRI	Low incidence of complications and effective in earlier stages of osteonecrosis; greater number of progenitor cells had superior outcomes	⁵⁸
BMNCs	DS (device)		Local injection	13	Osteonecrotic femoral head	Radiography MRI	1/10 hips in the BMNCs treated group showed progression to stage III compared to 5/8 hips in the control group after 24 months	⁵⁹
BMNCs	DS (device)		Local injection	60	Atrophic tibial nonunion	Radiography	Bone union obtained in 53 patients after an average of 12 weeks; bone formation correlated with MSCs number and concentration	⁴³
BMNCs	DS	PRP	β-TCP	1	Cleft lip and alveolus	CT	Sufficient bone regeneration shown after 6 months	¹⁰⁰
BMNCs	DS (device)		β-TCP ± local bone chips	41	Posterior spinal fusion	CT	Positive bony fusion was observed within 12 months postoperatively	¹⁹
BMNCs	SR (device)		Calcium phosphate	101	Bone healing disturbances	Radiology	No complications were reported and only 2/101 need further surgery within an average follow-up period of 14 months; bone formation not evaluated	¹⁰¹
BMNCs	SR (device)		Local injection	101	Bone healing disturbances	Radiology		¹⁰¹
BMNCs	Blood cell lysis		Local injection	342	Osteonecrotic femoral head	MRI	440/534 treated hips showed either complete resolution or a decreased osteonecrotic volume at an average follow-up duration of 12 years	⁶²
BMNCs	SR (device)		Collagen sponge	10	Volumetric bone deficiencies	Radiography	All patients showed bony healing and/or sufficient new bone formation within an average follow-up period of 8.3 months	¹⁰²
BMNCs	SR (device)		Allograft	3	Osteonecrotic femoral condyle	Radiography	Excellent graft integration and no further disease progression or subchondral collapse at 2 years	¹⁰³
BMNCs	SR (device)		Autograft	1	Atrophic mandibular fracture nonunion	Radiography	Excellent integration of the bone graft at 4 month follow-up	¹⁰⁴
BMNCs	SR (device)	PRP	Xenograft	26	Atrophied maxillary sinus	Histomorphometry	Biopsies obtained after an average of 3.41 months indicated no difference in new bone formation between BMNCs treatment and autografts	⁵³
BMNCs	DS		Collagen sponge, HA	39	Volumetric bone deficiencies	Radiography	New bone was detected in all patients whereas complete bone healing occurred in 36 cases within a follow-up period of 6 months; postoperative bone formation was significantly earlier in the HA compared to collagen treated group	⁵⁰
BMNCs	Magnetic sorting	PRP	Allograft	24	Distal tibial fractures	Radiography	All fractures healed within 12 month follow-up; significant faster fusion rate in the BMNCs treated compared to control group	⁵³
BMNCs	DS		Local injection	62	Osteonecrotic femoral head	Radiography CT	72/78 hips achieved a satisfactory clinical result while only 6 hips required replacement after a follow-up period of 5 years	⁶¹
BMNCs	DS		Allograft	18	Bone fracture nonunion	Radiography	All bone gaps were filled and faster healing in cell-based group compared to autografts	²¹
BMNCs	DS		β-TCP	10	Humerus fracture	Radiography	In all cases bone fracture healed within observation time of 12 weeks	²²
SVF	Centrifuge	PRP	Autografts	1	Calvarial defect	CT	Marked ossification in 120 cm² defect area 3 months after surgery	¹⁰⁵
SVF	Centrifuge	PRP	Hyaluronic acid	2	Osteonecrotic femoral head	MRI	Significant filling of bone defects 12 weeks after treatment	¹⁰⁶
SVF	Centrifuge	PRP	Hyaluronic acid	2	Osteonecrotic femoral head	MRI	Persistent bone regeneration sustained for at least 7 and 16 months after treatment	¹⁰⁷
SVF	Centrifuge	PRP	Hyaluronic acid	91	Osteonecrotic femoral head, osteoathritic knee/ankle, spinal disc herniation	Radiology	No neoplastic complications; bone formation not evaluated	⁵⁴
SVF	Unclear (device)		β-TCP, BCP	6	MSFE, dental implant placement	μCT and histology	Biopsies obtained 6 months after surgery showed higher bone volumes in SVF treated group compared to cell-free control, in particular in β-TCP treated patients	²⁹
SVF	Centrifuge	PRP	Silicated-HA	8	Humerus fracture	μCT and histology	In 5/6 biopsies de novo bone formation in the implant within 12 months	³⁰
ADMSCs	Filtration (device)		Allograft	8	Maxillary cysts and/or mandibular odontogenic	Histology	Biopsy obtained 3 months after treatment showed compact bone	⁵⁷
PB-derived CD34⁺ cells	Magnetic sorting		Atelocollagen, autograft	7	Femoral and tibial nonunion	Radiography	In 5/7 cases fracture healing was achieved after 12 weeks	⁴⁷

ADMSCs, adipose tissue-derived mesenchymal stem cells; CT, computed tomography; μCT, micro-computed tomography; MRI, magnetic resonance imaging.

One interesting observation from all clinical studies is that researchers used medical devices to intraoperatively isolate cells in half of the retrieved clinical studies. This would assumedly improve the reproducibility of intraoperatively prepared tissue-engineered bone constructs. Moreover, these isolation devices are closed systems and can be used in the operating room without cell laboratory support, which makes intraoperative preparation more feasible and applicable. To isolate BM-derived cells, DS was more frequently used than SR. Although these two methods have been compared in preclinical studies, their effect on bone formation has not been reported in any clinical study. For adipose tissue-derived cell isolates, tissue aspiration combined with a collagenase digestion step was generally used to isolate SVF. Only one study utilized a filtration device to obtain ADMSCs without enzymatic collagenase treatment.⁵⁷ With regard to scaffolds, auto-/allo-/xenografts (11 of 29 studies) were more frequently used in clinical compared to preclinical studies. Furthermore, bioceramics and collagen were the second most commonly used scaffolds in the retrieved clinical studies. When these two scaffolds were compared in the same condition, the postoperative bone formation appeared 7 weeks earlier and bone healing was 5 weeks faster for bioceramics versus collagen.⁵⁰ Cells without a scaffold, but with native fibrin, were applied in five studies^13,58–61; however, its efficacy was not compared to constructs with a scaffold. Notably, PRP was widely used in these clinical studies to act as a cell carrier as well as a cytokine reservoir.

Efficiency of Intraoperative Approach

The literature search results show that using “easily accessible cells” to intraoperatively prepare tissue-engineered bone constructs has been explored in numbers of pre- and clinical studies so far. These studies used different cells, isolation procedures and seeding strategies and led to dissimilar therapeutic effects. It is valuable to compare these studies to aid the future design of intraoperatively prepared tissue-engineered bone constructs. Based on the published data, we ask the following questions:

Does the intraoperative approach improve bone healing compared to acellular controls?

From 28 animal studies using an orthotopic model, 24 studies compared intraoperative tissue-engineered bone constructs to cell-free constructs. Among these, 12 studies used intraoperatively available sorted cells. Irrespective of the source (e.g., from bone marrow (BMNCs), adipose tissue (PSCs), or peripheral blood (PB-derived EPCs and ECs), these cells enhanced bone formation compared to cell-free constructs.^{12,14,17,18,22,25,45,49,62–65} In contrast, unsorted cells (e.g., BMACs, SVF, and PBMNCs) appeared inconclusive regarding their regenerative capacity. Fourteen studies demonstrated a promotive effect on bone healing,^{11,14,17,18,30,48,64–71} while 4 studies showed comparable bone healing compared to cell-free constructs.^18,62,72,73 This discrepancy may be associated with the cell density used to generate these constructs. A high density of desired cells (10⁶/cm³ and 10⁵/cm³) seeded on the scaffold generally led to bone formation, while a low density (10⁴/cm³) did not.^48,49 Still, the optimal number of seeded cells required in a graft site remains unknown.

Due to the difficulty to set up a control group in clinical cases, comparison of these intraoperatively prepared tissue-engineered bone constructs with cell-free constructs is not practical. Only three studies reported the comparison between SVF- and BMNCs-based with cell-free constructs in a clinical setting and demonstrated superior bone healing for SVF- and BMNCs-based constructs.^29,49,53 Two studies used autografts as controls and demonstrated comparable bone formation of constructs prepared with BM-derived cells.^21,52 Cell density similarly appears to play an important role in the clinical outcome as in animal studies, as indicated by the superior bone healing outcome for constructs with higher numbers of progenitor cells (6000 CFU-f/cm³).^13,58

Do intraoperatively prepared bone constructs perform superior to conventionally tissue-engineered constructs?

The aim of the intraoperative construct preparation is to overcome limitations of conventional cell-based bone tissue engineering for bone regeneration. However, how efficient the intraoperative approach is compared with the conventional approach is rarely reported. In all searched publications, only four studies compared therapeutic effects of intraoperatively prepared constructs to conventional tissue-engineered constructs (Table 4). All studies showed an inferior capacity of intraoperatively prepared constructs in promoting bone healing,^11,66,72,73 which indicates the necessity to improve the osteogenic potential of seeded cells. Besides, limitations of this comparison exist as none of these studies used human-derived cells and donor-matched comparison.

Table 4.

Comparison of Therapeutic Efficiency of Intraoperative Approach with Conventional Bone Tissue Engineering

Cell source	Scaffold	Seeding density	Sample size	In vivo time	Species and repair site	Evaluation method	Efficacy of bone formation	Reference
Rat BMACs, cultured BMSCs	HA/TCP	Unclear	6	8 weeks	Rat femoral defect	Histomorphometry	43.3% bone fill in cultured BMSCs compared to 17.2% n BMACs	¹¹
Equine SVF, cultured ADMSCs	Collagen sponges	5 × 10⁵/construct	6	12 weeks	Rat calvarial defect	CT	25% new bone in cultured ADMSCs compared to 10% in SVF	⁷²
Rabbit SVF, cultured ADMSCs	PLGA	1 × 10⁶/construct	5	8 weeks	Rabbit segmental ulna defect	μCT	50% new bone in cultured ADMSCs compared to 30% in SVF	⁶⁶
Goat SVF, cultured ADMSCs	PLCL	5 × 10⁶ SVF/construct	5	6 months	Goat spinal fusion	Radiography	43% fusion rate in ADMSCs compared to 30% in SVF	⁷³

BMSCs, bone marrow-derived mesenchymal cells; ADMSCs, adipose tissue-derived mesenchymal stem cells.

Is a sorted cell-based approach superior to an unsorted cell-based approach?

In the retrieved publications, both sorted and unsorted cell populations for intraoperative preparation of tissue-engineered bone constructs were used. The use of sorted homogeneous cells, such as ADMSCs and PSCs, facilitates improved standardization and greater control on the outcome of bone healing. Disadvantages include longer processing times and a requirement of extra instruments. In contrast, unsorted heterogeneous populations, such as BMNCs, SVF, and PBMNCs, contain multiple cell types (e.g., MSCs, ECs, pericytes, and monocytes) and may enable accelerated bone regeneration through heterotypic interactions among endogenous cell populations. The drawback of this method is the uncertain composition of the heterogeneous cell isolate. Of seven studies comparing the therapeutic efficiency of the sorted and unsorted cell-based intraoperative approach, six studies showed superior bone healing capacity of enriched cell isolates compared to constructs prepared using unsorted cells,^{20,25,44,45,64,74} except for one study that demonstrated similar bone healing results.¹⁵ This information further suggests the importance of cell enrichment during the intraoperative preparation of tissue-engineered bone constructs.

Which tissue-derived cells have superior bone healing effects for intraoperatively prepared tissue-engineered bone constructs?

Respecting the bone formation capacity of cells from different tissues, only one study compared effects of SVF (adipose tissue) with BMAC (bone marrow) and showed the superiority of BMAC over SVF regarding bone formation.⁶² Therefore, more comprehensive, donor-matched comparison is desired to draw a clear conclusion.

Limitations

The available preclinical and clinical data clearly show an enhanced regenerative effect of intraoperatively prepared tissue-engineered constructs to repair bone. However, bringing this approach into the realm of standard clinical care still requires overcoming several existing limitations. First, based on available reports, the therapeutic effectiveness of intraoperatively prepared tissue-engineered constructs is inferior to conventional cell-based bone constructs for treatment of bone defects. This is likely ascribed to the immature design and technique of intraoperative construct preparation as it is developed in a short period of time. Therefore, more controlled preclinical trials are necessary to optimize the preparation procedure and to improve the therapeutic effectiveness. Second, the balance between “effectiveness” and “practicality” of intraoperative construct preparation is merely considered in the previous studies. For instance, sorting cells by FACS has the potential to improve regenerative outcomes. However, utilization of this technique also requires higher number of cells, more operational steps, and longer preparation time, which render this approach less practical. Future studies should consider making the procedure compatible with both an intraoperative timeline and a reproducible and effective outcome. Furthermore, given the significant cell heterogeneity in most intraoperatively prepared tissue-engineered constructs, it is more difficult to elucidate the mechanism of bone healing with such constructs compared to conventional cell-based bone constructs. This issue becomes more serious when considering the donor variation in the clinic. Many factors contribute to such variability, including age, gender, medical condition (e.g., comorbidities such as osteoporosis and diabetes), and lifestyle (e.g., smoking). Consequently, there is a need to identify the key parameters for preparing successful intraoperatively prepared bone constructs, such as adequate cell numbers and good state of functional cells, before widespread application of this novel regenerative strategy.

Future Perspectives

According to the preclinical and clinical studies, further improvements in the therapeutic efficacy of intraoperatively prepared tissue-engineered bone constructs are suggested. We propose to take following strategies into consideration:

Enrichment of desired cells

Previous studies revealed a positive correlation between bone regeneration and the number of osteoprogenitors in BMNC-based constructs.^13,14 Except for the reported DS and SR methods to isolate BMNCs, cell sorting methods based on cell surface markers of specific cell types have been developed to select the target and to remove unwanted cell populations. For instance, a highly homogenous CD34⁺ cell population can be concentrated from PB in short time by FACS.⁴¹ This was also applied to select CD34⁺ MSCs from adipose tissue and exhibited five times more bone formation after 8 weeks compared to unsorted SVF cells.⁷⁵ With the same principle, magnetically activated cell sorting has been used to isolate BM-derived connective tissue progenitors on the basis of their hyaluronan antigens.²⁰ Based on this idea, an easier, inexpensive, and more feasible method was invented to use DNA aptamers, which are single-stranded DNA or RNA with high specificity and affinity to target cells.⁷⁶ Notably, these aptamers can be customized by labeling them with functional groups and retain their activities on scaffolds after implantation.⁷⁷ Of note, all these methods are premised on the basis of a well-defined marker panel of desired cells and the minor influence of immunoselection on the osteogenesis of sorted cells.^78,79

Coculture methods

Except for the interaction between heterogenetic cells in intraoperatively prepared constructs, whose mechanism and benefits on bone formation have not been well illustrated and proved, the reported cell–cell interactions between purified cells can be utilized to potentially improve bone healing. More importantly, this strategy can use intraoperatively available cells from different tissue(s) to fully take advantage of various cell types. For instance, cells from BM have high osteogenic potential, while CD34⁺ cells from fat tissue have high vascular potential. This gives rise to the possibility to use cells from BM and adipose tissue to generate intraoperatively prepared tissue-engineered vascularized bone constructs. Another example is PB-derived cell subsets, such as monocytes, macrophages,⁸⁰ regulatory T cells,^81,82 HCs,⁸³ and circulating EPCs. All these cells can be easily obtained and have proved to be involved in endogenous tissue healing and in certain cases can promote bone regeneration.^48,84 Therefore, these cell populations have the potential to improve bone healing when coseeded with cells from other tissue(s).

Scaffold design

To date, intraoperative preparation of tissue-engineered bone constructs is rather simplistic, typically seeding cells on the scaffold without control over cell composition, phenotype, and function in vivo. However, based on the aforementioned clinical evidence, bone healing following treatment with intraoperatively prepared tissue-engineered bone constructs depends on scaffold characteristics.^29,85 An ideal scaffold is able to concentrate and sort progenitor populations and to induce more rapid attachment of specific cell types during seeding, to promote cell survival and direct cell fate toward desired differentiation after seeding, and to improve bone and blood vessel formation after implantation. This design includes, but is not limited to, chemical, physical, and mechanical properties. Future technologies such as biomimetic engineering, 3D printing, and computational modeling will greatly aid the development of optimized scaffolds, which certainly requires a fundamental biological understanding of cell–material interactions.

Conclusion

From a practical view, intraoperative bone construct preparation will dramatically decrease the time, costs, and safety concerns and favor patient comfort. Cells from BM, adipose tissue, and PB provide the possibility to utilize this approach toward effective bone healing. Both preclinical and clinical studies displayed promising outcome when the intraoperative preparation concept was applied, and this concept will greatly benefit from new technologies, such as cell isolation devices, coculture method, and advanced fabrication of scaffolds. Collectively, the intraoperative approach demonstrates tremendous promise to advance stem cell therapies for bone repair and will serve as a new trend to generate cell-based regenerative constructs.

Footnotes

Acknowledgments

We acknowledge Dr. Sinan Guven's help in producing schematic representation of conventional bone tissue engineering. Funding: this study gets financial support from Dutch ZonMw funding (no. 40-41400-98–1401) and China Scholarship Council.

Disclosure Statement

No competing financial interests exist.

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