Research Paper
Mechanics of intact bone marrow

https://doi.org/10.1016/j.jmbbm.2015.06.023Get rights and content

Highlights

  • Bone marrow is a benign material with a large amount of intra- and inter-tissue heterogeneity.

  • The EEff of intact porcine marrow ranges from 0.3 to 24.7 kPa at physiological temperature.

  • Bulk rheology is the best method to capture the effects of temperature on tissue viscoelasticity.

  • Indentation is the ideal method for quantifying local tissue heterogeneity.

  • Cavitation mitigates destructive sample preparation, so tissue mechanics can be tested in situ.

Abstract

The current knowledge of bone marrow mechanics is limited to its viscous properties, neglecting the elastic contribution of the extracellular matrix. To get a more complete view of the mechanics of marrow, we characterized intact yellow porcine bone marrow using three different, but complementary techniques: rheology, indentation, and cavitation. Our analysis shows that bone marrow is elastic, and has a large amount of intra- and inter-sample heterogeneity, with an effective Young׳s modulus ranging from 0.25 to 24.7 kPa at physiological temperature. Each testing method was consistent across matched tissue samples, and each provided unique benefits depending on user needs. We recommend bulk rheology to capture the effects of temperature on tissue elasticity and moduli, indentation for quantifying local tissue heterogeneity, and cavitation rheology for mitigating destructive sample preparation. We anticipate the knowledge of bone marrow elastic properties for building in vitro models will elucidate mechanisms involved in disease progression and regenerative medicine.

Introduction

Bone marrow plays a significant role in body homeostasis by regulating immune and stromal cell trafficking. Researchers have characterized the matrix content and the role of local cells in bone physiology, but capturing the mechanics of bone marrow tissue has been limited in scope. The elastic modulus of engineered substrates is well known to influence cell shape, proliferation, migration, and differentiation (Marklein and Burdick, 2010, Peyton and Putnam, 2005, Peyton et al., 2008, Yang et al., 2014). While significant effort has gone into recapitulating the hematopoietic microenvironment in vitro for both regenerative medicine and to improve drug screening, there is no physiological measurement of the modulus of intact bone marrow (Lee et al., 2012, Mahadik et al., 2014, Nicholsa et al., 2010, Scotti et al., 2013, Torisawa et al., 2014). Though some of these model systems incorporate controlled mechanics, there is little validation for the stiffness choices, even though bone marrow stromal and progenitor cells are mechanically responsive to both engineered substrates, and the viscosity of the surrounding fluid (Engler et al., 2006, Lee et al., 2014, Sikavitsas et al., 2003, Yang et al., 2014). Knowing the modulus of in vivo tissue is critical for regenerative medicine as well. For example, the Blau lab found that the regenerative capacity of muscle stem cells is enhanced when cultured on surfaces mechanically similar to mouse muscle (Gilbert et al., 2010). This highlights the need for methods that can appropriately characterize the heterogeneous mechanics of bone marrow tissue to understand its role in driving the behaviors of the cells within.

Marrow tissue has hematopoietic-rich and adipose-rich regions, which are referred to as red and yellow marrow, respectively. Yellow marrow is enriched in the medullary cavity and red marrow in the spongy, trabecular bone (Parfitt et al., 1983, Vande Berg et al., 1998). Cell content in the marrow is a dynamic process, and yellow marrow can expand and contract as haematopoiesis occurs (Gimble et al., 1996). Unfortunately, the difficulty of harvesting red marrow has limited the ability to isolate and test its mechanics using conventional methods. Yellow marrow has been shown to be mechanically heterogeneous in studies where samples are homogenized and centrifuged to remove cell and bone debris (Bryant, 1988, Zhong and Akkus, 2011). Prepping samples in this manner removes many of the inconsistencies caused when harvesting marrow, but ignores the elastic contribution of the bone marrow extracellular matrix (ECM). The most robust study on yellow marrow mechanics measured the viscosity of the marrow from 19 human subjects and found no apparent correlation between age and marrow viscosity, though marrow has been shown to yellow with age (Justesen et al., 2001, Zhong and Akkus, 2011). Another group found proximal bovine marrow, the tissue close to the trabecular bone, to be more viscous than distal bovine marrow, and they suggest that these changes in viscosity are a function of spatial marrow composition (Bryant et al., 1988). Though both of these studies are informative, the impact of the surrounding or, potentially inclusive, trabecular bone is neglected because samples were homogenized and filtered.

The anatomical location and surrounding cortical bone poses a unique challenge for researchers interested in mechanically studying bone marrow tissue. Many studies have looked at properties of homogenized marrow, by extracting marrow from the medullary cavity and performing bulk rheology, but these approaches are destructive and create a critical gap in our knowledge of intact marrow mechanics (Bryant, 1988, Bryant et al., 1988, Saito et al., 2002, Sobotkova et al., 1988, Zhong and Akkus, 2011). In addition, researchers have used techniques to measure intramedullary pressure (IMP) to better understand how lifestyle choices, such as loading, disuse, steroid use, and diseases such as osteoporosis and cancer change marrow content, blood flow, and bone remodeling (Bloomfield, 2010, Gurkan and Akkus, 2008, Lynch et al., 2013, Miyanishi et al., 2002, Zhang et al., 2007). It is clear that many external factors impact IMP changes, but no work has gone into characterizing the mechanics of the intact matrix, which we suggest plays a stiffness-dependent role in disease progression. Rheology is the dominant method used to characterize bone marrow tissue, with the exception of one group that used ultrasonic wave propagation (Hosokawa and Otani, 1997). However, ultrasonic wave propagation reported that the bulk modulus of bovine marrow is the same order of magnitude as what others have found for the surrounding spongy bone (~2 GPa) (Morgan et al., 2003). The stark differences between marrow and bone likely make it hard to distinguish the marrow mechanics with type of technique. Though the viscoelasticity of bone marrow makes it ideal for bulk rheological characterization, this technique lacks the ability to measure microscopic level heterogeneities and often requires destructive sample preparation. Since bone marrow tissue varies across the length of the bone, is cell-rich, and is highly vascularized, we aimed to explore two more sensitive methods in parallel with traditional rheology: indentation and cavitation rheology. This approach enabled us to explore the continuity of diverse mechanical techniques and sample preparations on the characterization of marrow mechanical properties. Together, this information will allow the field of tissue engineering to further improve the understanding of marrow mechanics and to build more accurate in vitro models of marrow tissue.

Section snippets

In vitro sample preparation

Femurs from grass-fed large black Tamworth Cross pigs, 6–10 months old, were gathered from a local butcher, and mechanical testing was conducted within 2 h post-opening of the bone cavity. Indentation and rheology samples were gathered from a bone cut lengthwise down the femur, and tissue samples were biopsy punched out of the medullary cavity and stored in phosphate buffer solution (pH 7.4) for mechanical testing (Fig. 1a). The porcine bones were cut horizontally across the medullary cavity of

Three techniques were used to characterize the elastic modulus of bone marrow

We harvested intact marrow, with minimal post-mortem time, from 6 to 10 month old pigs. Pig was chosen as a model organism because of their anatomical similarity to humans, and their widespread use as sources for biological materials and as subjects for medical device testing (Meurens et al., 2012, Sullivan et al., 2001). Further, the pig model allowed us to examine tissue-scale mechanical heterogeneity absent of many convoluting factors, such as age, diet, and race. Rheology, indentation, and

Discussion

A goal of tissue engineering is to recapitulate key features of tissues in vitro in order to better understand in vivo phenomena and apply this toward directing tissue function. The mechanical properties of a cell׳s microenvironment have been shown to dictate the migration and differentiation of marrow-derived mesenchymal stem cells, but little research has been conducted on mechanically characterizing bone marrow tissue (Engler et al., 2006, Peyton et al., 2011, Yang et al., 2014). To fully

Conclusion

Here, we used three non-destructive approaches to mechanically characterize intact bone marrow. Across rheology, indentation, and cavitation we found that bone marrow is a benign viscoelastic material. Also, we are the first to report that marrow has dominant elastic contribution when the tissue is intact, and we feel this knowledge supports that components of the microenvironment, besides blood flow, may contribute to body homeostasis is a stiffness dependent manner. We also stress that bone

Acknowledgments

We are grateful to Michael Imburgia, Dr. Sami Fakhouri, and Shruti Rattan for technical assistance and for insightful conversations. We would also like to thank Lauren Barney, Elizabeth Brooks, and Dr. Sam Polio for useful advice in manuscript preparation. SRP is a Pew Biomedical Scholar supported by the Pew Charitable Trusts. SRP was supported by a faculty development award from Barry and Afsaneh Siadat. This work was funded by an NIH New Innovator award (1DP2CA186573-01) awarded to SRP, a

References (46)

  • P. Zhang et al.

    Knee loading dynamically alters intramedullary pressure in mouse femora

    Bone

    (2007)
  • Bloomfield, S.A. 2010. Disuse osteopenia. Curr. Osteoporos. Rep. 8, pp. 91–97,...
  • A.J. Booth et al.

    Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation

    Am. J. Respir. Crit. Care Med.

    (2012)
  • J. Bryant

    On the mechanical function of marrow in long bones

    Eng. Med

    (1988)
  • J. Bryant et al.

    Rheology of bovine bone Marrow

    Proc Inst. Mech. Eng.

    (1988)
  • E.P. Chan et al.

    Surface wrinkles for smart adhesion

    Adv. Mater.

    (2008)
  • S. Chatelin et al.

    Fifty years of brain tissue mechanical testing: from in vitro to in vivo investigations

    Biorheology

    (2010)
  • Davis, B.L., Praveen, S., 2006. Nonlinear versus linear behaviour of calcaneal bone marrow at different shear rates....
  • P. Gilbert et al.

    Substrate elasticity regulates skeletal muscle stem cell self- renewal in culture

    Science

    (2010)
  • U.A. Gurkan et al.

    The mechanical environment of bone marrow: a review

    Ann. Biomed. Eng.

    (2008)
  • A. Hosokawa et al.

    Ultrasonic wave propagation in bovine cancellous bone

    J. Acoust. Soc. Am.

    (1997)
  • S.B. Hutchens et al.

    Soft-solid deformation mechanics at the tip of an embedded needle

    Soft Matter

    (2014)
  • J. Justesen et al.

    Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis

    Biogerontology

    (2001)
  • Cited by (144)

    • Engineering the viscoelasticity of gelatin methacryloyl (GelMA) hydrogels via small “dynamic bridges” to regulate BMSC behaviors for osteochondral regeneration

      2023, Bioactive Materials
      Citation Excerpt :

      The stiffness of GelMA hydrogels has been found to regulate many cell behaviors including stem cell differentiation [23,31]. Then, in order to further elucidate the dissipative properties of GelMA hydrogels on the effect of BMSC behaviors, three viscoelastic hydrogels were prepared (for which the ratios of loss modulus to storage modulus were 0.02, 0.21, and 0.32; see Fig. S11 and Table S2) with a similar level of storage moduli (G' ∼ 4 kPa), which was similar to that of marrow [66]. Before cell culture, the porosity, swelling behavior, degradation of hydrogels and their effect on the mechanical stability were carefully explored.

    • Injectable bone marrow microniches by co-culture of HSPCs with MSCs in 3D microscaffolds promote hematopoietic reconstitution from acute lethal radiation

      2023, Bioactive Materials
      Citation Excerpt :

      Therefore, we optimized the Young's modulus, mechanical properties (e.g., viscoelasticity), degradability and injectability of the microscaffolds to meet the requirements of intramedullary injection, thereby directly promoted HSCs homing and colonization to the BM. In accordance with the Young's modulus (0.3–65 kPa) and internal pore size (20–100 μm) of natural BM structure [14,15], the Young's modulus and internal pore size of the microscaffolds were 11.2 kPa and 84 μm, respectively (Fig. 4a, b, c). We observed that the crosslinking degree (45.8% vs. 9.5%), the storage modulus (1.94 kPa vs. 0.12 kPa), and the loss modulus (98.4 Pa vs. 9.87 Pa) of injectable microscaffolds were significantly lower than that of non-injectable microscaffolds (Fig. 4d, e, f).

    View all citing articles on Scopus
    View full text