Application of Decellularized ECM in Bone and Cartilage Tissue

Engineering

Although synthetic materials can be modified to simulate the mechanical and biochemical
properties of the cell microenvironment, these materials fall short of fully replicating the
tissue’s microenvironment. One potential therapeutic material, that can be used to improve
tissue regeneration, is the native extracellular matrix (ECM), which is the non-cellular
component of tissue that provides the structural support and biochemical cues for determining
a cell's fate. ECM is a natural material that encompasses both the cell microenvironment and
biochemical factors for living cells. It contains a reservoir of growth factors and cytokines; these
send signals that regulate cell proliferation and migration as well as modulate differentiation
and phenotypic expression of the cell.

Bone ECM consists of an organic and inorganic phase. The organic phase, mostly type I collagen,
provides the tissue with flexibility, while the inorganic phase, mainly consisting of calcium
phosphate, specifically hydroxyapatite (HA), is the source of bone strength.

Cartilage ECM is primarily a collagenous network, with varying compositions and types of
collagen depending on the cartilage type. Hyaline cartilage is mainly type II collagen, while
fibrous cartilage is a mixture of both type I and II collagens. Another major component of these
networks is proteoglycans. Proteoglycans consist of multiple chains of glycosaminoglycans
(GAGs) branching off from a core protein.

Decellularization
Decellularization refers to the process of treating tissue with any combination of i) Physical
stress, ii) Chemical and iii) Enzymatic agents to remove cellular components, leaving behind
only the non-cellular ECM that can be used for therapeutic applications.

The main benefit of decellularized ECM (dECM) is that it retains components of the natural cell
environment. With proper decellularization, the complex biomolecular and physical cues in the
ECM are preserved and can support cell growth and viability. Unlike in transplanted tissue,
dECM has a lower risk for immune response because almost all the cellular DNA is removed. To
retain as much of the tissue's bioactivity as possible while maximizing the removal of nuclear
material, the decellularization process must minimize the loss of native ECM components.
Implantation of decellularized tissue that has had its nucleic materials incompletely removed or
degraded could result in host foreign body reaction, which leads to the formation of fibrous
capsule surrounding the implant site.
Physical Decellularization
Freeze-thawing is one of the most widely used physical decellularization methods, during which
the formation of ice crystals puncture cell membranes. The cycle is repeated multiple times
before the tissue sample can be processed further.
Another option is osmotic lysis, during which tissues are placed in either a hypertonic or
hypotonic solution such as deionized water that ruptures the plasma membrane via osmotic
shock. Other common physical decellularization methods include hydrostatic pressure,
sonication, and electroporation.
Tissues that undergo only physical decellularization, specifically freeze-thawing, are considered
to be devitalized but not decellularized, as the cells have been lysed, but the cell debris and
genetic material still remain within the processed tissue. Devitalized tissue particles have been
shown to have higher quantities of ECM components, such as GAGs, than those that have been
additionally treated with chemical/ enzymatic decellularization methods. However, there are
safety concerns of possible immune responses that could result from residual cellular material.

Chemical Decellularization
Chemical methods of decellularization can largely be divided into two subcategories where
tissue samples can be treated with either (a) acidic or basic conditions or (b) detergents.

Treating tissues with acids or bases results in cell degradation and the removal of cellular
components such as nucleic acids. The degree of successful decellularization will vary according
to the type and concentration of the acid/ base being used, processing time, and the type of
tissue being treated. Bases are considered the harsher option of the two and can result in
significant loss of GAGs. Preservation of GAGs during decellularization is important to maintain
tissue mechanical properties and to retain growth factors in the tissue.

Chemical decellularization can also be performed through the use of detergents. Three main
types of detergents are used: nonionic, ionic, and zwitterionic. Nonionic detergents such as
Triton X-100 lyse cells through insertion into the lipid bilayer, disrupting the cellular membrane.
While disrupting lipid interactions, these largely preserve protein–protein interactions. Proteins
are solubilized, but their native structure mostly remains intact.
Ionic detergents such as sodium dodecyl sulfate (SDS) are known as denaturing detergents;
they disrupt cell membranes and also completely denature proteins. Ionic detergents are
considered harsher than nonionic detergents, and they are more detrimental to the ECM
structure.
Zwitterionic detergents result in less denaturing of proteins compared to ionic detergents, they
also tend to remove less cellular material than ionic detergents.
Enzymatic Decellularization

Nucleases and proteases are the most widely used enzymes for enzymatic decellularization.
Nucleases, such as deoxy ribonuclease and ribonuclease, act directly on DNA and RNA chains,
respectively, to hydrolyze phosphodiester bonds. Proteases, such as trypsin, act on proteins by
hydrolyzing peptide bonds.
Enzymatic methods are frequently used in conjunction with chelating agents such as
ethylenediaminetetraacetic acid (EDTA), which disrupt cell adhesion to ECM proteins by
sequestering metallic ions such as calcium.

Physical decellularization is the least disruptive decellularization method, with most of the ECM
components and structure left intact after treatment. However, physical decellularization alone
cannot completely remove cellular debris from the tissue. Often it is used in conjunction with
additional chemical or enzymatic methods.
Similarly, incubating a tissue sample in chemical or enzymatic agents without physical agitation
does not result in an acceptable degree of decellularization due to limited diffusion into the
tissue. Therefore, a combination of all three methods has a synergistic effect where physical
agitation enhances the tissue penetration depth of chemical and enzymatic agents, thereby
facilitating the removal of lysed cell material.

Decellularized ECM as Scaffold

One of the simplest methods of using dECM is as a scaffold that maintains its original geometry.
The biggest advantage of this method is that using it as an unprocessed scaffold suggests that
the tissue retains a large portion of its original ECM architecture. dECM can be prepared from
various tissue types to accommodate different compositions, topographies, and mechanical
properties. However, such benefits can only be obtained if (a) most of the cell debris is
removed from the tissue without destruction of essential ECM components such as GAGs and
collagen fibers and (b) the dECM can be thoroughly recellularized.

MSCs are seeded on decellularized bones from donors that expresses osteogenic markers.
Decellularized bone has also been subjected to further modifications, such as collagen/HA
coating. When type I collagen solutions were applied to the surface of decellularized porcine
cancellous bone, the coating modulated the stiffness of the matrix.

Luo et al. introduced channels into full-thickness porcine cartilage discs, which acted as
conduits for fluids and cells to penetrate into the tissue. These channels supported cell viability
and attachment while also allowing the cells to align with the native collagen architecture.
Organ Reengineering through Development of a Transplantable
Recellularized Liver Graft using Decellularized Liver Matrix

Decellularization is an attractive technique for scaffold preparation in tissue engineering, as the

resulting material can potentially retain the architecture of the original tissue, including the
functional aspects of the native microvasculature.

In this work, they modified and applied perfusion decellularization technique to prepare whole
liver grafts, and introduced perfusion-seeding and culture techniques for the preparation of
recellularized liver matrix for transplantation.

A key advantage of this methodology is the preservation of liver-specific extracellular matrix

and three-dimensional architecture, providing crucial cues for hepatocyte engraftment, survival
and long-term function. Most importantly, the decellularized liver matrix (DLM) has the
underlying matrix of the vascular network, which can be readily connected to the circulation,
facilitating rapid oxygen and nutrient delivery after transplantation.

Decellularization of Liver Matrix

Liver decellularization was achieved by portal perfusion with SDS that lyses cells and solubilizes
cytoplasmic components. After 72 hours of decellularization, a translucent acellular scaffold,
which retained the gross shape of liver, was generated. 100% of the fibrillary collagen and
approximately 50% of the GAG of native liver were retained after decellularization.
The vast majority of the smaller microcirculatory branches were preserved, indicating that
physiologic flow could be achieved by traversing the portal venous system and emptying into
systemic venous circulation via the hepatic vein and inferior vena cava.

Recellularization of Decellularized Liver Matrix

Roughly 12.5 million cells were introduced at each step, for a total of four steps, with 10
minutes intervals between each step. After seeding the DLM with 50 million cells, the
recellularized liver grafts were transferred into a specially designed perfusion chamber for in
vitro culture.
The perfusion chamber features two hermetically sealed silicon sheets, forming a pouch filled
with culture medium; this design avoids rigid surfaces, preventing development of pressure
spots, while enabling sterile culture of the recellularized grafts up to 2 weeks in vitro.
Scalability of Recellularization Methodology

Reconstruction of liver grafts in vitro requires the addition of non-parenchymal cells. To allow
endothelial cell engraftment, a non-parenchymal component was seeded to the recellularized
liver graft by incorporating “microvascular endothelial cells” to the recellularized graft. Analysis
showed that endothelial cells were capable of lining the vasculature encircled by hepatocytes.

Transplantation of Recellularized Liver Grafts

Recipient rats underwent removal of liver to prepare a viable site for auxiliary liver graft
transplantation. To create blood flow within the graft renal vein and ports were used as ports.
The graft was quickly filled with blood as the artery was unclamped. The recellularized graft was
kept in vivo for 8 hours and it was observed that there was minimal damage to hepatocytes due
to blood flow and resulting shear stress. But the hepatic function was retained in the
transplanted graft with minimal ischemic damage.

So, after decellularization, the preservation of liver’s three dimensional architecture, functional
vasculature and native matrix composition were observed. Then Recellularization was done
which maintained cell viability and function. Finally, the recellularized graft was transplanted
and it was functional with a little ischemic damage.

Online Methods

Liver Decellularization

Frozen liver was thawed at 4oC and washed with PBS (Phosphate Buffer Saline) overnight
through perfusion via portal vein. Liver was fused with SDS and distilled water in 3 steps for 5
days. Then the liver was washed with distilled water and Triton X-100 for 45 minutes to remove
residual SDS. Finally the decellularized liver was washed with PBS for another one hour.

Hepatocyte Seeding

The Decellularized Liver Matrix (LDM) was connected to the perfusion system through portal
vein cannulation. Inferior and Superior vena cava wee left open as outlet. DLM was kept
immersed in medium in a perfusion chamber while it was perfused through the portal vein.
After a 30-min perfusion with medium, a total of 50 or 200 million primary adult rat
hepatocytes with over 90% viability were infused into the circuit in four steps at 10-min
intervals and were recirculated in the system at 25 °C.
Recellularized Liver Graft in Perfusion System

For perfusion culture experiments, the recellularized liver was transferred to a clean chamber.
It consisted of a peristaltic pump, bubble trap and oxygenator. The graft was continuously
perfused through the portal vein at 15 ml min−1 with continuous oxygenation that delivered an
inflow partial oxygen tension of ~300 mm Hg.

Heterotropic Recellularized Graft Transplantation

The liver graft was connected with the renal artery and vein of the recipient rat. The graft was
placed in the left renal space after the left ureter was ligated and transected. Saline containing
heparin and a glycoprotein inhibitor was injected into penile vein to prevent coagulation.

Two stents were introduced into donor’s portal vein and inferior vena cava. Donor’s portal vein
and renal artery of receiver were connected with two sutures. The donor’s inferior vena cava
were sutured in the left renal vein of the receiver. Then the arterial and venous clamps were
removed to start perfusion.
Microfluidic Endothelium for Studying the intravascular Adhesion of
Metastatic Breast Cancer Cell

Specific interactions between circulating cancer cells and vascular endothelium are proposed to
control patterns of metastasis for breast, lung, and other common solid cancers.
Identifying molecular determinants of trafficking and arrest of circulating cancer cells on
endothelium at characteristic sites of metastatic disease has been limited in large part by
challenges of studying the intravascular microenvironment under physiologic conditions.
In vitro assays of cancer cells and endothelium typically are performed under static,
biochemically homogeneous conditions. These static assays do not accurately model
intravascular events in metastasis because blood flow alters gene expression, mechanical
properties of cells, and cell adhesion.

So, a microfluidic model of the vasculature has been engineered to overcome these limitations
and to undertake an advance study of the intravascular compartment in cancer metastasis. This
microfluidic device produces defined flow rates within a range of physiologic levels and enables
real-time bright field and fluorescence imaging of circulating cancer cells and microvascular
endothelium. This also mimics serial interaction of circulating tumor cells with endothelia of
differing potentials to promote cell adhesion and formation of metastases.

Effect of chemokine CXCL12, receptor CXCR4, CXCR7 on adhesion of circulating breast cancer
cells to endothelium was also investigated. It was observed that high level of CXCL12 are
expressed by parenchymal cells in organs affected by metastatic breast cancer, such as liver,
bone and brain. That is, gradients of CXCL12 regulate homing of disseminated breast cancer
cells to malignant breast, lung and other cancer cells for characteristic sites of metastatic
disease has been attributed predominantly to signaling through receptor CXCR4 on tumor cells.

Fabrication of Microfluidic Device

The microfluidic vasculature is comprised of two PDMS layers sandwiching a thin, porous, and
optically clear polyester membrane. The top channel contains a confluent monolayer of human
dermal microvascular endothelial cells (HDMECs) cultured on the polyester membrane with 400
nm pores to permit diffusion of biomolecules between the top and bottom channels.

By activating endothelia over only one of the regions, the device reproduces serial interactions
of tumor cells with endothelia of differing metastasis-supporting potential as occur
physiologically. This setup enables simultaneous and distinct localization of chemokines on the
basal face and flow on the apical face of the endothelium.
Region Selective Treatment of the Microfluidic Endothelium

Circulating “231-control” breast cancer cells showed greater adhesion onto the regions of the
endothelium treated with cytokine TNF-a compared to the untreated regions. As the shear
stress was increased from 0.5dyn cm-2 to 2.5dyn cm-2, the adhesion selectivity towards TNF-a
regions became greater.

Adhesion of Cancer Cells Stably Expressing CXCR4 or CXCR7 onto Microfluidic

Endothelium

Three cancer cells, 231-control, 231-CXCR4 (231-control expressing CXCR4) and 231-CXCR7
(231-control expressing CXCR7) were compared to assess effects of CXCL12 chemokine
receptors on cancer cells in mediating adhesion onto microfluidic endothelium.

For each of the three cancer cell types, adhesion preference was towards CXCL12-treated
endothelium over the corresponding untreated endothelium in the same device. The adhesion
of 231-CXCR4 or 231-CXCR7 were found to be greater than 231-control cells towards CXCL12-
treated endothelium. But the effect of CXCL12 treatment were statistically same for all three
types of cells. So, CXCL12-dependent enhancement in adhesion is independent of expression of
CXCR4 or CXCR7 in cancer cells.

CXCR4 on Endothelial Cells Mediates Adhesion of Cancer Cells

CXCL12 upregulated CXCR4 in HDMECs and TNF-a upregulated both CXCR4 and CXCR7. And
AMD3100 is an inhibitor of CXCL12 binding to CXCR4. Now, to assess the contribution of CXCR4
in cancer cell binding, they region selectively treated this endothelium with various
combinations of CXCL12, TNF-a and AMD3100. 231-control cell was used which do not express
CXCR4 or CXCR7.
Now, there was preferential adhesion to the treated endothelium for all four of the treatment
combinations except for CXCL12+AMD3100. This shows that the enhanced adhesion selectivity
of cancer cells is due to CXCL12 signaling through CXCR4 on the vascular endothelium.

This system possesses unique blend of extravascular stimulation of vascular endothelium with
CXCL12 and region-specific endothelium stimulation with chemokines. This helps to compare
cancer cell adhesion to endothelium of differing metastatic potential within the same
experiment. It has also been shown that TNF-a elicits greater leukocyte trans-endothelial
migration across endothelium treated from the basal versus apical side which suggests that
spatial localization of adhesion molecules may be influenced by directionality of cytokine
stimulation.
Using the microfluidic endothelium, we confirmed that CXCR4 or CXCR7 on breast cancer cells
promotes intravascular adhesion throughout the channel, supporting a mechanism through
which these receptors expressed on cancer cells promote metastasis.
Comparison of Human Induced Pluripotent and Embryonic Stem Cells

Human induced pluripotent stem cells (hiPSCs) have been hailed as an effective replacement
for human embryonic stem cells (hESCs) and a prime candidate cell source for regenerative
medicine aims. Both hESCs and hiPSCs share the important properties of self-renewal and
pluripotency; that is, they are theoretically capable of generating unlimited amounts of any
differentiated cell in the human body.

hESCs are derived from the inner cell mass of fresh or frozen embryos at the blastocyst stage of
development. hESCs self-renew to allow for indefinite maintenance of the undifferentiated
state in vitro and thereby retain the ability to differentiate into derivatives of the three
embryonic germ layers that subsequently form all the tissues of a developing fetus.
Consequently, hESCs are a promising candidate cell source for the generation of differentiated
cells for use in cell replacement therapies, as well as a valuable tool for disease modeling and
drug screening applications.
Unfortunately, however, hESC derivation remains ethically controversial in the United States
and somewhat challenging logistically because of a limited supply of donor human embryos.

In contrast to hESCs, hiPSCs are derived by “reprogramming” of somatic cells to a pluripotent

state through the overexpression of a key set of transcription factors. This process does not
require the destruction of human embryos ex utero, thereby circumventing much of the ethical
debate surrounding hESC derivation. In addition, because the techniques for hiPSC derivation
are easily applicable to adult somatic cell types, cell lines can be easily derived from a variety of
genetic backgrounds. This allows not only for the creation of patient-specific hiPSCs that are
theoretically secure against immune rejection.

hiPSCs are similar to hESCs in terms of their morphology, feeder dependence, surface marker
expression, and in vivo teratoma formation capacity. Although global gene expression profiles
of hESCs and hiPSCs are largely similar, subtle differences in the expression of messenger RNAs
(mRNAs) and micro RNAs have been reported.

Some of the differences between hiPSCs and hESCs appear to be related to the hiPSC’s somatic
cell of origin in the form of an “epigenetic memory,” a term that refers to persisting epigenetic
marks from the cell type of origin in the resulting hiPSC that continue to affect gene expression.

Deviation of hiPSC from ESC

First, although most genes exhibit similar degrees of variation in hiPSC and hES lines, a small
number of genes exhibited substantially increased deviation from the hESC reference standard
in hiPSCs. Only a very small fraction of this gene expression variation was attributable to
epigenetic memory of the somatic cell of origin.
Second, hESCs and hiPSCs can be thought of as two overlapping clouds in which some, but not
all, hiPSCs can be distinguished from hESCs. However, no unique epigenetic or transcriptional
deviation was found to be shared by all tested hiPSC lines.

Third, heterogeneity among single cells in hESC populations in vitro has previously been shown
to underlie important cell fate decisions, with initial evidence suggesting an increased degree of
heterogeneity among single hiPSCs than among single hESCs.

Although initial comparisons on a global scale revealed considerable similarity between hESCs
and hiPSCs, closer inspection at finer resolution reveals differences. If related cell types are
thought of as analogous to siblings, hESCs and hiPSCs can perhaps be thought of as twins.
Identical twins are much more difficult to distinguish from each other, but there are
appreciable differences upon closer inspection. For hESCs and hiPSCs, whether these
differences are functionally consequential or simply related to the scale of analysis remains
largely unknown.

The epigenetic marks that set hiPSCs apart from hESCs carry an unfairly negative connotation,
because epigenetic memory can be used judiciously to bias hiPSCs toward a cell fate of interest.
The ability to prime PSCs selectively toward the desired cell lineage—using epigenetic memory,
cytokines, genetic modification, small molecules, or otherwise—may therefore occupy
increasing interest in the future.
Methods for Making Induced Pluripotent Stem Cells

Self-renewal and pluripotency are defining properties of embryonic stem cells (ESCs). They
refer, respectively, to the ability to proliferate indefinitely without com-mitment in vitro and to
the capacity to differentiate into cell lineages belonging to the three embryonic germ layers.

ESCs are derived from the inner cell mass (ICM) of pre-implantation embryos. An alternative
way, ‘reprogramming’ — represents a simple way to obtain pluripotent stem-cell lines from
almost any somatic tissue and mammalian species. The use of such cells also circumvents the
ethical issues associated with human ESCs.

Reprogramming needs the in trans-expression in a somatic cell of a set of core pluripotency-

related transcription factors [in most cases OCT4, SOX2, KLF4 and MYC (OSKM)]. When
successful, tightly compacted colonies appear on the culture dish; these colonies resemble ESCs
morphologically, molecularly and phenotypically.
These cells are called induced Pluripotent Stem Cells (iPSCs). iPSCs are relevant to a range of
applications, including: autologous cell therapy; the modelling of monogenic and multigenic
diseases; the study of complex genetic traits and allelic variation; and as substrates for drug,
toxicity, differentiation and therapeutic screens.

Variables of Reprogramming

Reprogramming is an extremely slow and inefficient process influenced by several variables

that affect its efficiency, reproducibility and the quality of the resulting iPSCs. Before choosing a
reprogramming approach it is therefore important to identify these variables. Some of these
variables are discussed below:

Donor Cell Type

Depending on the donor cell type, reprogramming is achieved with different efficiencies and
kinetics. For example, 8–12 days are required to reprogramme mouse embryonic fibroblasts
(MEFs) using retroviruses, whereas the same process takes 20–25 days for human foreskin
fibroblasts (HFFs). Again, compared with fibroblasts, human primary keratinocytes transduced
with OSKM are reprogrammed 100 times more efficiently and twofold faster.

The differentiation status of the starting cell type also affects reprogramming efficiency. For
example, haematopoietic stem and progenitor cells generate 300 times more iPSC colonies
than do terminally differentiated B and T cells.
Reprogramming Cocktail (Pluripotency+ Cell Proliferation + Epigenetics)

Pluripotency: After the cell type is chosen, other reprogramming factors need to be selected.
Many of these factors are ‘genes’ that are normally expressed during development and
involved in the maintenance of pluripotency of the embryo. OCT4, SOX2 and NANOG are the
core pluripotency transcription factors.
When NANOG is expressed along with OSKM in mouse B cells, the time until colony appearance
is reduced by half compared with that taken by OSKM alone. Again, when UTF1, another
pluripotency transcription factor, is expressed with OSKM in human primary fibroblasts, more
colonies with high levels of alkaline phosphatase are generated.

Cell Proliferation: Factors like MYC and KLF4 directly or indirectly affect cell proliferation. TERT
and SV40LT increase the appearance of ESC-like colonies when combined with OSKM.
MicroRNAs (miRNA) are also known to influence cell proliferation. Introduction of miR-294,
miR-295 into OCT4 of Mouse Embryonic Fibroblast (MEF) increases the number of colonies
compared with OSKM alone.

Epigenetics: Chemical compounds that alter DNA methylation or chromatin modification

improve reprogramming in various cell types. HDAC inhibitors improves reprogramming in
MEFs. Vitamin C also significantly improves the reprogramming of MEFs and adult mammary
gland fibroblasts, in part by alleviating cell senescence and inducing DNA demethylation.

Culture Conditions

Culture conditions, supportive cells and medium compositions all parameters have effect on
reprogramming efficiency.
Reprogramming under hypoxic conditions of 5% O2, instead of the atmospheric 21% O2,
increases the reprogramming efficiency of mouse and human cells by 40- and fourfold,
respectively. When combined with VPA, the efficiency increases to 200-fold in mouse cells.
Supportive feeder cells secrete growth factors that are required for ESC survival and
proliferation and inhibition of ESC spontaneous differentiation.

Delivery Systems

Viral Delivery Method: The delivery of OSKM transcription factors into fibroblast was originally
done using MMLV-derived retroviruses. The vector, in which the reprogramming cDNA is
cloned, provides a viral packaging signal and transcription elements.
Lentiviral delivery vectors have also been successfully used to express different sets pf
reprogramming factors in somatic cells.
Although they are efficient and reproducible, reprogramming using viruses entails the
production of potentially harmful viral particles that express potent oncogenes such as MYC.
Moreover, they unavoidably generate heterogeneous iPSC lines, which could complicate
comparative analysis. Even if properly silenced, viral transgenes can eventually be reactivated
during differentiation or during the life of iPSC-derived or transplanted animals, leading to
tumors.

Non-Viral Delivery Method: No transgene or vector is used during this method. This approach
addresses the limitations like, permanent genetic modification resulting from the integration of
retroviral or lentiviral vectors. RNA delivery, protein delivery, episomal delivery are different
approaches.
But this process is slower than the previous one. Reprogramming vary between different
starting cell types and species, the generation of stable iPSCs usually requires several weeks to
complete. One of the major drawbacks in this approach is that they are ususally inefficient and
poorly reproducible.

It is still difficult to choose a reprogramming strategy that is fitting for all purposes. In the first
situation, the reprogramming approach needs to be robust and efficient method and
combination of factors. The second case, on the other hand, requires a non-integrative
approach in order to avoid or control genomic modification. The use of ‘safe’ approaches does
not necessarily prevent variability in the expression levels of lineage-specification genes or the
occurrence of aberrant epigenetic remodeling.

To improve reprogramming efficiency and kinetics, small molecules can be used. Small
molecules represent a powerful alternative or support for reprogramming because they can
target different cellular pathways that control cell fate, state and function, but their specificity
is sometimes difficult to assess.